Production of peracids using an enzyme having perhydrolysis activity

ABSTRACT

A process is provided for producing peroxycarboxylic acids from carboxylic acid esters. More specifically, carboxylic acid esters are reacted with an inorganic peroxide, such as hydrogen peroxide, in the presence of an enzyme catalyst having perhydrolysis activity. The present perhydrolase catalysts are classified as members of the carbohydrate esterase family 7 (CE-7) based on the conserved structural features. Further, disinfectant formulations comprising the peracids produced by the processes described herein are provided.

This application is a DIV of U.S. patent application Ser. No.11/743,354, filed May 2, 2007, now U.S. Pat. No. 7,951,566, which is acontinuation-in-part of U.S. patent application Ser. No. 11/638,635,filed Dec. 12, 2006, now U.S. Pat. No. 7,964,378 which claims thebenefit of U.S. Provisional Application No. 60/750,092, filed Dec. 13,2005, and U.S. Provisional Application No. 60/853,065, filed Oct. 20,2006.

FIELD OF THE INVENTION

This invention relates to the field of peracid biosynthesis and in situenzyme catalysis. Specifically, a process is provided to produceperacids using the perhydrolysis activity of enzymes identifiedstructurally as belonging to the CE-7 family of carbohydrate esterases,including cephalosporin acetyl hydrolases (CAHs; E.C. 3.1.1.41) andacetyl xylan esterases (AXEs; E.C. 3.1.1.72). The enzymatic processproduces percarboxylic acids from carboxylic acid ester substrates.Further, disinfectant formulations comprising the peracids produced bythe processes described herein are provided.

BACKGROUND OF THE INVENTION

Peracid compositions have been reported to be effective antimicrobialagents. Methods to clean, disinfect, and/or sanitize hard surfaces, meatproducts, living plant tissues, and medical devices against undesirablemicrobial growth have been described (U.S. Pat. No. 6,545,047; U.S. Pat.No. 6,183,807; U.S. Pat. No. 6,518,307; U.S. patent applicationpublication 20030026846; and U.S. Pat. No. 5,683,724). Peracids havealso been reported to be useful in preparing bleaching compositions forlaundry detergent applications (U.S. Pat. No. 3,974,082; U.S. Pat. No.5,296,161; and U.S. Pat. No. 5,364,554).

Peracids can be prepared by the chemical reaction of a carboxylic acidand hydrogen peroxide (see Organic Peroxides, Daniel Swern, ed., Vol. 1,pp 313-516; Wiley Interscience, New York, 1971). The reaction is usuallycatalyzed by a strong inorganic acid, such as concentrated sulfuricacid. The reaction of hydrogen peroxide with a carboxylic acid is anequilibrium reaction, and the production of peracid is favored by theuse of an excess concentration of peroxide and/or carboxylic acid, or bythe removal of water. There are several disadvantages to the chemicalreaction for peracid production: a) the high concentration of carboxylicacid used to favor production of peracid can result in an undesirableodor when using the peracid-containing solution, 2) the peracid isoftentimes unstable in solution over time, and the concentration ofperacid in the solution decreases during storage prior to use, and 3)the formulation is often strongly acidic due to the use of aconcentrated sulfuric acid as catalyst.

One way to overcome the disadvantages of the chemical production ofperacids is to employ an enzyme catalyst in place of a strong acidcatalyst. The use of an enzyme catalyst allows for the rapid productionof peracid at the time of use and/or application, avoiding problemsassociated with storage of peracid solutions and variations in peracidconcentrations over time. The high concentrations of carboxylic acidstypically used to produce peracid via the direct chemical reaction withhydrogen peroxide are not required for enzymatic production of peracid,where the enzyme-catalyzed reaction can use a carboxylic acid ester assubstrate at a much lower concentration than is typically used in thechemical reaction. The enzyme reaction can be performed across a broadrange of pH, dependent on enzyme activity and stability at a given pH,and on the substrate specificity for perhydrolysis at a given pH.

Esterases, lipases, and some proteases have the ability catalyze thehydrolysis of alkyl esters to produce the corresponding carboxylic acids(Formula 1).

Some esterases, lipases, and proteases also exhibit perhydrolysisactivity, catalyzing the synthesis of peracids from alkyl esters(Formula 2).

O. Kirk et al. (Biocatalysts, 11:65-77 (1994)) investigated the abilityof hydrolases (lipases, esterases, and proteases) to catalyzeperhydrolysis of acyl substrates with hydrogen peroxide to formperoxycarboxylic acids, and reported that perhydrolysis proceeds with avery low efficiency in aqueous systems. Furthermore, they found thatlipases and esterases degraded percarboxylic acid to the correspondingcarboxylic acid and hydrogen peroxide. They also found that proteasesneither degraded nor catalyzed perhydrolysis of carboxylic acid estersin water. The authors concluded that esterases, lipases and proteasesare, in general, not suitable for catalyzing perhydrolysis of simpleesters, such as methyl octanoate and trioctanoin, in an aqueousenvironment.

U.S. Pat. No. 3,974,082 describes the production of bleachingcompositions for laundry detergent applications by contacting thematerial to be bleached with an aqueous solution containing anoxygen-releasing inorganic peroxygen compound, an acyl alkyl ester, andan esterase or lipase capable of hydrolyzing the ester.

U.S. Pat. No. 5,364,554 describes an activated oxidant system for insitu generation of peracid in aqueous solution using a protease enzyme,a source of hydrogen peroxide, and an ester substrate that is preferablychemically non-perhydrolyzable. A method of bleaching and a method offorming peracid are also disclosed.

U.S. Pat. No. 5,296,161 describes production of peracid in an aqueoussolution comprising one or more specific esterases and lipases, a sourceof hydrogen peroxide, and a functionalized ester substrate suitable foruse in a bleaching composition. However, the concentration of peracidproduced was generally insufficient for use in many commercialdisinfectant applications.

Most known methods for preparing peracids from the correspondingcarboxylic acid esters using enzyme catalysts do not produce andaccumulate a peracid at a sufficiently-high concentration to beefficacious for disinfection in a variety of applications. Severalprotease and lipase combinations have recently been reported to generateperacids (e.g., peracetic acid) in situ at concentrations suitable foruse as a disinfectant and/or commercial bleaching agent (see co-ownedU.S. patent application Ser. Nos. 11/413,246 and 11/588,523; hereinincorporated by reference). However, there remains a need to identifyadditional perhydrolase catalysts capable of producing peracids in situ.

U.S. Pat. No. 4,444,886 describes a strain of Bacillus subtilis (ATCC31954™) having ester hydrolase activity (described as a “diacetinase”)that has high specificity for hydrolyzing glycerol esters having acylgroups having 2 to 8 carbon atoms. U.S. Pat. No. 4,444,886 does notdescribe, discuss or predict that the ester hydrolase activity of thisstrain has perhydrolase activity towards carboxylic acid esters,including glycerol esters.

The problem to be solved is to provide a process to enzymaticallyproduce peracids in situ at concentrations suitable for use in a varietyof disinfectant applications and/or bleaching applications. Preferably,the substrates used to produce the peracid compositions should berelatively non-toxic and inexpensive, such as carboxylic acid esters,especially mono-, di-, and triacylglycerols, where the acyl group has1-8 carbon atoms, as well as acetylated sugars.

SUMMARY OF THE INVENTION

The stated problems have been solved by the discovery that enzymesbelonging to the structural family of CE-7 esterases (e.g.,cephalosporin C deacetylases [CAHs] and acetyl xylan esterases [AXEs])exhibit significant perhydrolysis activity for converting the presentcarboxylic acid esters (in the presence of an inorganic source ofperoxygen such as hydrogen peroxide) into peracids at concentrationssufficient for use as a disinfectant and/or bleaching agent. The systemachieves efficiency by producing the peracid in high concentrationswithout requiring a high concentration of peroxygen.

Specific examples of perhydrolases are exemplified from Bacillussubtilis (ATCC 31954™), B. subtilis BE1010 (Payne and Jackson, J.Bacteriol. 173:2278-2282 (1991)), B. subtilis ATCC 6633™ (U.S. Pat. No.6,465,233), B. subtilis ATCC 29233™; B. licheniformis ATCC 14580 μm (Reyat al., Genome Biol., 5(10):article 77 (2004)), Clostridium thermocellumATCC 27405™ (Copeland at al., GENBANK® ZP_(—)00504991, B. pumilus PS213(Degrassi et al., Microbiology, 146:1585-1591 (2000)), and Thermotoganeapolitana (GENBANK® AAB70869.1).

Each of the present perhydrolases described herein share conservedstructural features (i.e. a conserved signature motif) as well assuperior perhydrolysis activity when compared to other α/β-hydrolases,(such as commercially available lipases; see comparative Examples 26 and28), making this unique family of enzymes particularly suitable forgenerating peracids in situ at concentrations sufficient for use as adisinfectant and/or bleaching agent. Suitable perhydrolases useful inthe present process can be identified by a conserved signature motiffound within the CE-7 family of carbohydrate esterases.

In one aspect of the invention, a process is provided, including aprocess for producing a peroxycarboxylic acid from a carboxylic acidester comprising

-   -   a) providing a set of reaction components, said components        comprising:        -   1) a carboxylic acid ester selected from the group            consisting of:            -   i) esters having the structure

-   -   -   -   wherein R₁=C1 to C7 straight chain or branched chain                alkyl optionally substituted with an hydroxyl or a C1 to                C4 alkoxy group and R₂═C1 to C10 straight chain or                branched chain alkyl, alkenyl, alkynyl, aryl, alkylaryl,                alkylheteroaryl, heteroaryl, (CH₂CH₂—O)_(n)H or                (CH₂CH(CH₃)—O)_(n)H and n=1 to 10; and            -   ii) glycerides having the structure

-   -   -   -   wherein R₁═C1 to C7 straight chain or branched chain                alkyl optionally substituted with an hydroxyl or a C1 to                C4 alkoxy group and R₃ and R₄ are individually H or                R₁C(O); and            -   iii) acetylated saccharides selected from the group                consisting of acetylated monosaccharides, acetylated                disaccharides, and acetylated polysaccharides;

        -   2) a source of peroxygen; and

        -   3) an enzyme catalyst having perhydrolysis activity, wherein            said enzyme catalyst comprises an enzyme comprising a            signature motif when aligned to a reference sequence SEQ ID            NO: 2 using CLUSTALW, said signature motif comprising:            -   i) an RGQ motif at amino acid positions 118-120;            -   ii) a GXGSG motif at amino acid positions 179-183; and            -   iii) an HE motif at amino acid positions 298-299; and

    -   b) combining said reaction components under suitable aqueous        reaction conditions whereby a peroxycarboxylic acid is produced.

In another aspect of the invention, a process is provided, including aprocess to disinfect a hard surface or inanimate object using anenzymatically-produced peroxycarboxylic acid composition, said processcomprising:

-   -   a) providing a set of reaction components, said components        comprising:        -   1. a substrate selected from the group consisting of:            -   i) esters having the structure

-   -   -   -   wherein R₁═C1 to C7 straight chain or branched chain                alkyl optionally substituted with an hydroxyl or a C1 to                C4 alkoxy group and R₂═C1 to C10 straight chain or                branched chain alkyl, alkenyl, alkynyl, aryl, alkylaryl,                alkylheteroaryl, heteroaryl, (CH₂CH₂—O)_(n)H or                (CH₂CH(CH₃)—O)_(n)H            -   and n=1 to 10; and            -   ii) glycerides having the structure

-   -   -   -   wherein R₁═C1 to C7 straight chain or branched chain                alkyl optionally substituted with an hydroxyl or a C1 to                C4 alkoxy group and R₃ and R₄ are individually H or                R₁C(O);            -   iii) acetylated saccharides selected from the group                consisting of acetylated monosaccharides, acetylated                disaccharides, and acetylated polysaccharides;

        -   2) a source of peroxygen; and

        -   3) an enzyme catalyst having perhydrolysis activity, wherein            said enzyme catalyst comprises a signature motif when            aligned to a reference sequence SEQ ID NO: 2 using CLUSTALW,            said signature motif comprising:            -   i) an RGQ motif at amino acid positions 118-120;            -   ii) a GXGSG motif at amino acid positions 179-183; and            -   iii) an HE motif at amino acid positions 298-299; and

    -   b) combining said reaction components under suitable aqueous        reaction conditions whereby a peroxycarboxylic acid product is        formed;

    -   c) optionally diluting said peroxycarboxylic acid product; and

    -   d) contacting said hard surface or inanimate object with the        peroxycarboxylic acid produced in step b) or step c) whereby        said surface or said inanimate object is disinfected.

In another aspect of the invention, a system is provided, including aperoxycarboxylic acid generating system comprising:

a) a substrate selected from the group consisting of:

-   -   1) esters having the structure

-   -   wherein R₁=C1 to C7 straight chain or branched chain alkyl        optionally substituted with an hydroxyl or a C1 to C4 alkoxy        group and R₂═C1 to C10 straight chain or branched chain alkyl,        alkenyl, alkynyl, aryl, alkylaryl, alkylheteroaryl, heteroaryl,        (CH₂CH₂—O)_(n)H or (CH₂CH(CH₃)—O)_(n)H and n=1 to 10; and    -   2) glycerides having the structure

-   -   wherein R₁═C1 to C7 straight chain or branched chain alkyl        optionally substituted with an hydroxyl or a C1 to C4 alkoxy        group and R₃ and R₄ are individually H or R₁C(O); and    -   3) acetylated saccharide selected from the group consisting of        acetylated monosaccharides, acetylated disaccharides, and        acetylated polysaccharides; and

b) a source of peroxygen; and

c) an enzyme catalyst having perhydrolysis activity, wherein said enzymecatalyst comprises an enzyme having a signature motif when aligned to areference sequence SEQ ID NO: 2 using CLUSTALW, said signature motifcomprising:

-   -   i) an RGQ motif at amino acid positions 118-120;    -   ii) a GXGSG motif at amino acid positions 179-183; and    -   iii) an HE motif at amino acid positions 298-299.

In another aspect of the invention, a formulation is provided, saidformulation comprising:

-   -   a) a first mixture comprising an enzyme catalyst comprising a        perhydrolase enzyme having a CE-7 signature motif and an        carboxylic acid ester selected from the group consisting of        monoacetin, diacetin, triacetin and mixtures thereof; said first        mixture optionally comprising a further component selected from        the group consisting of an inorganic or organic buffer, a        corrosion inhibitor, a wetting agent, and combinations thereof;        and    -   b) a second mixture comprising a source of peroxygen and water,        said second mixture optionally further comprising a chelating        agent.

In another aspect of the invention, a formulation is provided, saidformulation comprising:

a) a first mixture comprising a enzyme catalyst comprising aperhydrolase enzyme having a CE-7 signature motif and an acetylatedsaccharide selected from the group consisting of acetylatedmonosaccharides, acetylated disaccharides, acetylated polysaccharides,and combinations thereof, said first mixture optionally furthercomprising an inorganic or organic buffer, a corrosion inhibitor, and awetting agent; and

b) a second mixture comprising a source of peroxygen and water, saidsecond mixture optionally comprising a chelating agent.

In other aspects the first and second mixtures above may be combined toprovide for production of peroxycarboxylic acid. In a further aspect,the first and second mixtures above may be combined to form disinfectantformulations.

Another aspect of the invention is a recombinant Escherichia coli cellcomprising a disruption in katE and a disruption in katG with theproviso that the host cell is not Escherichia coli UM2. In anotheraspect, the recombinant Escherichia coli host cell comprising adisruption in katE and a disruption in katG, is derived from Escherichiacoli strain MG1655. In another aspect, the Escherichia coli host cell orstrain produces or comprises at least one enzyme having perhydrolaseactivity. In another aspect, said enzyme is capable of using orgenerating hydrogen peroxide.

The enzyme catalyst having perhydrolysis activity used in the presentprocess may be in the form of non-viable whole cells, permeabilizedwhole cells, one or more cell components of a microbial cell extract,partially-purified enzyme, and purified enzyme. The enzyme catalyst maybe unimmobilized or immobilized, including but not limited to:immobilization in or on an insoluble solid support, covalently attachedto a soluble polymer (e.g., low-molecular weight polyethylene glycol(PEG)), and immobilized as soluble enzyme in a hollow-fiber cartridge.

In another aspect of the invention, a method is provided to reduce aconcentration of a microbial population on a hard surface or inanimateobject by contacting the peracid composition produced by the aboveprocesses with said hard surface or inanimate object, whereby theconcentration of the viable microbial population is reduced at least3-log, preferably at least 4-log, more preferably at least 5-log, andmost preferably at least 6-log. In a further aspect, the peracidcomposition produced by the above methods may be optionally diluted to adesired efficacious concentration prior to contacting the surface orinanimate object to be treated.

BRIEF DESCRIPTION OF THE FIGURE

FIG. 1 (panels a-c) is a CLUSTALW alignment of perhydrolases of thepresent invention. Each of the perhydrolases are structurally classifiedmembers of the carbohydrate esterase family 7 (CE-7) and share certainconserved domains. Several conserved motifs (underlined) have beenidentified that together form the signature motif for CE-7 carbohydrateesterases. An additional motif (LXD; amino acid residues 267-269 of SEQID NO: 2) is also underlined and may be used to further characterize thesignature motif.

BRIEF DESCRIPTION OF THE BIOLOGICAL SEQUENCES

The following sequences comply with 37 C.F.R. 1.821-1.825 (“Requirementsfor Patent Applications Containing Nucleotide Sequences and/or AminoAcid Sequence Disclosures—the Sequence Rules”) and are consistent withWorld Intellectual Property Organization (WIPO) Standard ST.25 (1998)and the sequence listing requirements of the European Patent Convention(EPC) and the Patent Cooperation Treaty (PCT) Rules 5.2 and 49.5(a-bis), and Section 208 and Annex C of the Administrative Instructions.The symbols and format used for nucleotide and amino acid sequence datacomply with the rules set forth in 37 C.F.R. §1.822.

A Sequence Listing is provided herewith on Compact Disk. The contents ofthe Compact Disk containing the Sequence Listing are hereby incorporatedby reference in compliance with 37 CFR 1.52(e).

SEQ ID NO: 1 is the nucleic acid sequence of the cephalosporin Cdeacetylase (cah) coding region from Bacillus subtilis ATCC 31954™.

SEQ ID NO: 2 is the deduced amino acid sequence of the cephalosporin Cdeacetylase from Bacillus subtilis ATCC 31954™.

SEQ ID NOs: 3 and 4 are primers used to PCR amplify perhydrolase genesfrom Bacillus subtilis species for construction of pSW186, pSW187,pSW188, and pSW190.

SEQ ID NO: 5 is the nucleic acid sequence of the cephalosporin Cdeacetylase coding region from B. subtilis subsp. subtilis str, 168.

SEQ ID NO: 6 is the deduced amino acid sequence of the cephalosporin Cdeacetylase from B. subtilis subsp. subtilis str. 168, and is identicalto the deduced amino acid sequence of the cephalosporin C deacetylasefrom B. subtilis BE1010.

SEQ ID NO: 7 is the nucleic acid sequence of the cephalosporinacetylesterase coding region from B. subtilis ATCC 6633™.

SEQ ID NO: 8 is the deduced amino acid sequence of the cephalosporinacetylesterase from B. subtilis ATCC 6633™.

SEQ ID NO: 9 is the nucleic acid sequence of the cephalosporin Cdeacetylase coding region from B. licheniformis ATCC 14580™.

SEQ ID NO: 10 is the deduced amino acid sequence of the cephalosporin Cdeacetylase from B. licheniformis ATCC 14580™.

SEQ ID NO: 11 is the nucleic acid sequence of the acetyl xylan esterasecoding region from B. pumilus PS213.

SEQ ID NO: 12 is the deduced amino acid sequence of the acetyl xylanesterase from B, pumilus PS213.

SEQ ID NO: 13 is the nucleic acid sequence of the acetyl xylan esterasecoding region from Clostridium thermocellum ATCC 27405™.

SEQ ID NO: 14 is the deduced amino acid sequence of the acetyl xylanesterase from Clostridium thermocellum ATCC 27405™.

SEQ ID NO: 15 is the nucleic acid sequence of the acetyl xylan esterasecoding region from Thermotoga neapolitana.

SEQ ID NO: 16 is the deduced amino acid sequence of the acetyl xylanesterase from Thermotoga neapolitana.

SEQ ID NO: 17 is the nucleic acid sequence of the acetyl xylan esterasecoding region from Thermotoga maritima MSB8.

SEQ ID NO: 18 is the deduced amino acid sequence of the acetyl xylanesterase from Thermotoga maritima MSB8.

SEQ ID NO: 19 is the nucleic acid sequence of the acetyl xylan esterasecoding region from Thermoanaerobacterium sp. JW/SL YS485.

SEQ ID NO: 20 is the deduced amino acid sequence of the acetyl xylanesterase from Thermoanaerobacterium sp. JW/SL YS485.

SEQ ID NO: 21 is the nucleic acid sequence of the cephalosporin Cdeacetylase coding region from Bacillus sp. NRRL B-14911.

SEQ ID NO: 22 is the deduced amino acid sequence of the cephalosporin Cdeacetylase from Bacillus sp. NRRL B-14911.

SEQ ID NO: 23 is the nucleic acid sequence of the cephalosporin Cdeacetylase coding region from Bacillus halodurans C-125.

SEQ ID NO: 24 is the deduced amino acid sequence of the cephalosporin Cdeacetylase from Bacillus halodurans C-125.

SEQ ID NO: 25 is the nucleic acid sequence of the cephalosporin Cdeacetylase coding region from Bacillus clausii KSM-K16.

SEQ ID NO: 26 is the deduced amino acid sequence of the cephalosporin Cdeacetylase from Bacillus clausii KSM-K16.

SEQ ID NOs: 27 and 28 are primers used to PCR amplify perhydrolase genesfrom Bacillus subtilis species for construction of pSW194 and pSW189.

SEQ ID NO: 29 is the nucleic acid sequence of the PCR product clonedinto pSW194.

SEQ ID NO: 30 is the nucleic acid sequence of the PCR product clonedinto pSW189.

SEQ ID NO: 31 is the nucleic acid sequence of the Bacillus subtilis ATCC29233™ cephalosporin C deacetylase (cah) gene cloned into pSW190.

SEQ ID NO: 32 is the deduced amino acid sequence of the Bacillussubtilis ATCC 29233™ cephalosporin C deacetylase (CAH).

SEQ ID NOs: 33 and 34 are primers used to PCR amplify the Bacilluslicheniformis ATCC 14580™ cephalosporin C deacetylase gene forconstruction of pSW191.

SEQ ID NOs: 35 and 36 are primers used to PCR amplify the Clostridiumthermocellum ATCC 27405™ acetyl xylan esterase gene for construction ofpSW193.

SEQ ID NOs: 37 and 38 are primers used to PCR amplify the Bacilluspumilus PS213 acetyl xylan esterase coding sequence (GENBANK® AJ249957)for construction of pSW195.

SEQ ID NOs: 39 and 40 are primers used to PCR amplify the Thermotoganeapolitana acetyl xylan esterase gene (GENBANK® 58632) for constructionof pSW196.

SEQ ID NO: 41 is the nucleic acid sequence of the codon-optimizedversion of the Thermotoga neapolitana acetyl xylan esterase gene inplasmid pSW196.

SEQ ID NO: 42 is the nucleic acid sequence of the kanamycin resistancegene.

SEQ ID NO: 43 is the nucleic acid sequence of plasmid pKD13.

SEQ ID NOs: 44 and 45 are primers used to generate a PCR productencoding the kanamycin gene flanked by regions having homology to thekatG catalase gene in E. coli MG1655. The product was used to disruptthe endogenous katG gene.

SEQ ID NO: 46 is the nucleic acid sequence of the PCR product encodingthe kanamycin resistance gene flanked by regions having homology to thekatG catalase gene in E. coli MG1655. The product was used to disruptthe endogenous katG gene.

SEQ ID NO: 47 is the nucleic acid sequence of the katG catalase gene inE. coli MG1655.

SEQ ID NO: 48 is the deduced amino acid sequence of the KatG catalase inE. coli MG1655.

SEQ ID NO: 49 is the nucleic acid sequence of plasmid pKD46.

SEQ ID NOs: 50 and 51 are primers used to confirm the disruption of thekatG gene.

SEQ ID NO: 52 is the nucleic acid sequence of plasmid pCP20.

SEQ ID NO: 53 and 54 are primers used to generate a PCR product encodingthe kanamycin gene flanked by regions having homology to the katEcatalase gene in E. coli MG1655. The product was used to disrupt theendogenous katE gene.

SEQ ID NO: 55 is the nucleic acid sequence of the PCR product encodingthe kanamycin resistance gene flanked by regions having homology to thekatE catalase gene in E. coli MG1655. The product was used to disruptthe endogenous katE gene.

SEQ ID NO: 56 is the nucleic acid sequence of the katE catalase gene inE. coli MG1655.

SEQ ID NO: 57 is the deduced amino acid sequence of the KatE catalase inE. coli MG1655.

SEQ ID NOs: 58 and 59 are primers used to confirm disruption of the katEgene in the single knockout strain E. coli MG1655 ΔkatE, and in thedouble-knockout strain E. coli MG1655 ΔkatG ΔkatE, herein referred to asE. coli KLP18.

SEQ ID NO: 60 is the nucleic acid sequence of the codon optimizedversion of the Bacillus pumilus PS213 encoding the amino acid sequenceSEQ ID NO: 12.

SEQ ID NO: 61 is the amino acid sequence of the region encompassingamino acids residues 118 through 299 of SEQ ID NO: 2.

DETAILED DESCRIPTION OF THE INVENTION

The stated problems have been solved by the discovery that enzymesbelonging to the CE-7 carbohydrate esterase family exhibit significantperhydrolysis activity for converting carboxylic acid ester substratesto peracids. This family of structurally related enzymes can be used togenerate concentrations of peracids with high efficiency fordisinfection and/or bleaching applications.

In this disclosure, a number of terms and abbreviations are used. Thefollowing definitions apply unless specifically stated otherwise.

As used herein, the term “comprising” means the presence of the statedfeatures, integers, steps, or components as referred to in the claims,but that it does not preclude the presence or addition of one or moreother features, integers, steps, components or groups thereof.

As used herein, the term “about” modifying the quantity of an ingredientor reactant of the invention or employed refers to variation in thenumerical quantity that can occur, for example, through typicalmeasuring and liquid handling procedures used for making concentrates oruse solutions in the real world; through inadvertent error in theseprocedures; through differences in the manufacture, source, or purity ofthe ingredients employed to make the compositions or carry out themethods; and the like. The term “about” also encompasses amounts thatdiffer due to different equilibrium conditions for a compositionresulting from a particular initial mixture. Whether or not modified bythe term “about”, the claims include equivalents to the quantities.

As used herein, the term “peracid” is synonymous with peroxyacid,peroxycarboxylic acid, peroxy acid, percarboxylic acid and peroxoicacid.

As used herein, the term “peracetic acid” is abbreviated as “PAA” and issynonymous with peroxyacetic acid, ethaneperoxoic acid and all othersynonyms of CAS Registry Number 79-21-0.

As used herein, the term “monoacetin” is synonymous with glycerolmonoacetate, glycerin monoacetate, and glyceryl monoacetate.

As used herein, the term “diacetin” is synonymous with glyceroldiacetate; glycerin diacetate, glyceryl diacetate, and all othersynonyms of CAS Registry Number 25395-31-7.

As used herein, the term “triacetin” is synonymous with glycerintriacetate; glycerol triacetate; glyceryl triacetate,1,2,3-triacetoxypropane, 1,2,3-propanetriol triacetate and all othersynonyms of CAS Registry Number 102-76-1.

As used herein, the term “monobutyrin” is synonymous with glycerolmonobutyrate, glycerin monobutyrate, and glyceryl monobutyrate.

As used herein, the term “dibutyrin” is synonymous with glyceroldibutyrate and glyceryl dibutyrate.

As used herein, the term “tributyrin” is synonymous with glyceroltributyrate, 1,2,3-tributyrylglycerol, and all other synonyms of CASRegistry Number 60-01-5.

As used herein, the term “monopropionin” is synonymous with glycerolmonopropionate, glycerin monopropionate, and glyceryl monopropionate.

As used herein, the term “dipropionin” is synonymous with glyceroldipropionate and glyceryl dipropionate.

As used herein, the term “tripropionin” is synonymous with glyceryltripropionate, glycerol tripropionate, 1,2,3-tripropionylglycerol, andall other synonyms of CAS Registry Number 139-45-7.

As used herein, the term “ethyl acetate” is synonymous with aceticether, acetoxyethane, ethyl ethanoate, acetic acid ethyl ester, ethanoicacid ethyl ester, ethyl acetic ester and all other synonyms of CASRegistry Number 141-78-6.

As used herein, the term “ethyl lactate” is synonymous with lactic acidethyl ester and all other synonyms of CAS Registry Number 97-64-3.

As used herein, the terms “acetylated sugar” and “acetylated saccharide”refer to mono-, di- and polysaccharides comprising at least one acetylgroup, Examples include, but are not limited to glucose pentaacetate,xylose tetraacetate, acetylated xylan, acetylated xylan fragments,β-D-ribofuranose-1,2,3,5-tetraacetate, tri-O-acetyl-D-galactal, andtri-O-acetyl-glucal.

As used herein, the terms “suitable enzymatic reaction mixture”,“components suitable for in situ generation of a peracid”, “suitablereaction components”, and “suitable aqueous reaction mixture” refer tothe materials and water in which the reactants and enzyme catalyst comeinto contact. The components of the suitable aqueous reaction mixtureare provided herein and those skilled in the art appreciate the range ofcomponent variations suitable for this process. In one embodiment, thesuitable enzymatic reaction mixture produces peracid in situ uponcombining the reaction components. As such, the reaction components maybe provided as a multicomponent system wherein one or more of thereaction components remains separated until use. The design of systemsand means for separating and combining multiple active components areknown in the art and generally will depend upon the physical form of theindividual reaction components. For example, multiple active fluids(liquid-liquid) systems typically use multichamber dispenser bottles ortwo-phase systems (U.S. Patent Application Pub. No. 2005/0139608; U.S.Pat. No. 5,398,846; U.S. Pat. No. 5,624,634; U.S. Pat. No. 6,391,840;E.P. Patent 0807156B1; U.S. Patent Appln. Pub. No. 2005/0008526; and PCTPublication No. WO 00/11713A1) such as found in some bleachingapplications wherein the desired bleaching agent is produced upon mixingthe reactive fluids. Other forms of multicomponent systems used togenerate peracid may include, but are not limited to those designed forone or more solid components or combinations of solid-liquid components,such as powders (e.g., many commercially available bleachingcomposition, U.S. Pat. No. 5,116,575), multi-layered tablets (U.S. Pat.No. 6,210,639), water dissolvable packets having multiple compartments(U.S. Pat. No. 6,995,125) and solid agglomerates that react upon theaddition of water (U.S. Pat. No. 6,319,888). In one embodiment, aformulation is provided as two individual mixtures whereby aperoxycarboxylic acid disinfectant is generated upon combining the twomixtures. In another embodiment, a formulation is provided comprising:

-   -   a) a first mixture comprising:        -   i) an enzyme catalyst having perhydrolase activity, said            enzyme catalyst comprising an enzyme having a CE-7 signature            motif; and        -   ii) a carboxylic acid ester substrate, said first mixture            optionally comprising a component selected from the group            consisting of an inorganic or organic buffer, a corrosion            inhibitor, a wetting agent, and combinations thereof; and    -   b) a second mixture comprising a source of peroxygen and water,        said second mixture optionally comprising a chelating agent.        In another embodiment, the carboxylic acid ester in the first        mixture is selected from the group consisting of monoacetin,        diacetin, triacetin, and combinations thereof. In another        embodiment, the carboxylic acid ester in the first mixture is an        acetylated saccharide. In another embodiment, the enzyme        catalyst in the first mixture is a particulate solid. In another        embodiment, the first reaction mixture is a solid tablet or        powder.

As used herein, the term “perhydrolysis” is defined as the reaction of aselected substrate with peroxide to form a peracid. Typically, inorganicperoxide is reacted with the selected substrate in the presence of acatalyst to produce the peracid. As used herein, the term “chemicalperhydrolysis” includes perhydrolysis reactions in which a substrate (aperacid precursor) is combined with a source of hydrogen peroxidewherein peracid is formed in the absence of an enzyme catalyst.

As used herein, the term “perhydrolase activity” refers to the catalystactivity per unit mass (for example, milligram) of protein, dry cellweight, or immobilized catalyst weight.

As used herein, “one unit of enzyme activity” or “one unit of activity”or “U” is defined as the amount of perhydrolase activity required forthe production of 1 μmmol of peracid product per minute at a specifiedtemperature.

As used herein, the terms “enzyme catalyst” and “perhydrolase catalyst”refer to a catalyst comprising an enzyme having perhydrolysis activityand may be in the form of a whole microbial cell, permeabilizedmicrobial cell(s), one or more cell components of a microbial cellextract, partially purified enzyme, or purified enzyme. The enzymecatalyst may also be chemically modified (e.g., by pegylation or byreaction with cross-linking reagents). The perhydrolase catalyst mayalso be immobilized on a soluble or insoluble support using methodswell-known to those skilled in the art; see for example, Immobilizationof Enzymes and Cells; Gordon F. Bickerstaff, Editor; Humana Press,Totowa, N.J., USA; 1997. As described herein, all of the present enzymeshaving perhydrolysis activity are structurally members of thecarbohydrate family esterase family 7 (CE-7 family) of enzymes (seeCoutinho, P. M., Henrissat, B. “Carbohydrate-active enzymes: anintegrated database approach” in Recent Advances in CarbohydrateBioengineering, H. J. Gilbert, G. Davies, B. Henrissat and B. Svenssoneds., (1999) The Royal Society of Chemistry, Cambridge, pp. 3-12.).

Members of the CE-7 family include cephalosporin C deacetylases (CAHs;EC. 3.1.1.41) and acetyl xylan esterases (AXEs; E.C. 3.1.1.72). Membersof the CE-7 esterase family share a conserved signature motif (Vincentet al., J. Mol. Biol., 330:593-606 (2003)). Perhydrolases comprising theCE-7 signature motif and/or a substantially similar structure aresuitable for use in the present invention. Means to identifysubstantially similar biological molecules are well known in the art(e.g. sequence alignment protocols, nucleic acid hybridizations,presence of a conserved signature motif, etc.). In one aspect, theenzyme catalyst in the present process comprises a substantially similarenzyme having at least 40%, preferably at least 50%, more preferably atleast 60%, even more preferable at least 70%, even more preferably atleast 80%, yet even more preferable at least 90% identity, and mostpreferably at least 95% amino acid identity to the sequences providedherein. The nucleic acid molecules encoding the present CE-7carbohydrate esterases are also provided herein. In a furtherembodiment, the perhydroiase catalyst useful in the present process isencoded by a nucleic acid molecule that hybridizes stringent conditionsto one of the present nucleic acid molecules.

As used herein, the terms “cephalosporin C deacetylase” and“cephalosporin C acetyl hydrolase” refers to an enzyme (E.C. 3.1.1.41)that catalyzes the deacetylation of cephalosporins such as cephalosporinC and 7-aminocephalosporanic acid (Mitsushima et al., supra). Asdescribed herein, several cephalosporin C deacetylases are providedhaving significant perhydrolysis activity.

As used herein, “acetyl xylan esterases”” refers to an enzyme (E.C.3.1.1.72; AXEs) that catalyzes the deacetylation of acetylated xylansand other acetylated saccharides. As illustrated herein, several enzymesclassified as acetyl xylan esterases are provided having significantperhydrolase activity.

As used herein, the term “Bacillus subtilis (ATCC 31954™)” refers to abacterial cell deposited to the American Type Culture Collection (ATCC)having international depository accession number ATCC 31954T″. Bacillussubtilis ATCC 31954™ has been reported to have an ester hydrolase(“diacetinase”) activity capable of hydrolyzing glycerol esters having 2to 8 carbon acyl groups, especially diacetin (U.S. Pat. No. 4,444,886;herein incorporated by reference in its entirety). As described herein,an enzyme having significant perhydrolase activity has been isolatedfrom B, subtilis ATCC 31954™ and is provided as SEQ ID NO: 2. The aminoacid sequence of the isolated enzyme has 100% amino acid identity to thecephalosporin C deacetylase provided by GenBank® Accession No.BAA01729.1.

As used herein, the term “Bacillus subtilis BE1010” refers to the strainof Bacillus subtilis as reported by Payne and Jackson (J. Bacteriol.173:2278-2282 (1991)). Bacillus subtilis BE1010 is a derivative Bacillussubtilis subsp. subtilis strain BR151 (ATCC33677™) having a chromosomaldeletion in the genes encoding subtilisin and neutral protease. Asdescribed herein, an enzyme having significant perhydrolase activity hasbeen isolated from B. subtilis BE1010 and is provided as SEQ ID NO: 6.The amino acid sequence of the isolated enzyme has 100% amino acididentity to the cephalosporin C deacetylase reported in Bacillussubtilis subsp. subtilis strain 168 (Kunst et al., supra).

As used herein, the term “Bacillus subtilis ATCC 29233™” refers to astrain of Bacillus subtilis deposited to the American Type CultureCollection (ATCC) having international depository accession numberATCC31954™. As described herein, an enzyme having significantperhydrolase activity has been isolated and sequence from B. subtilisATCC 29233™ and is provided as SEQ ID NO: 32.

As used herein, the term “Clostridium thermocellum ATCC 27405™” refersto a strain of Clostridium thermocellum deposited to the American TypeCulture Collection (ATCC) having international depository accessionnumber ATCC27405™. The amino acid sequence of the enzyme havingperhydrolase activity from C. thermocellum ATCC 27405™ is provided asSEQ ID NO: 14.

As used herein, the term “Bacillus subtilis ATCC6633™” refers to abacterial cell deposited to the American Type Culture Collection (ATCC)having international depository accession number ATCC6633™. Bacillussubtilis ATCC 6633™ has been reported to have cephalosporinacetylhydrolase activity (U.S. Pat. No. 6,465,233). The amino acidsequence of the enzyme having perhydrolase activity from B. subtilisATCC 6633™ is provided as SEQ ID NO: 8.

As used herein, the term “Bacillus licheniformis ATCC 14580™” refers toa bacterial cell deposited to the American Type Culture Collection(ATCC) having international depository accession number ATCC 14580™.Bacillus licheniformis ATCC 14580™ has been reported to havecephalosporin acetylhydrolase activity. The amino acid sequence of theenzyme having perhydrolase activity from B. licheniformis ATCC 14580™ isprovided as SEQ ID NO: 10.

As used herein, the term “Bacillus pumilus PS213” refers to a bacterialcell reported to have acetyl xylan esterase activity (GENBANK®AJ249957). The amino acid sequence of the enzyme having perhydrolaseactivity from Bacillus pumilus PS213 is provided as SEQ ID NO: 12.

As used herein, the term “Thermotoga neapolitana” refers to a strain ofThermotoga neapolitana reported to have acetyl xylan esterase activity(GENBANK® 58632). The amino acid sequence of the enzyme havingperhydrolase activity from Thermotoga neapolitana is provided as SEQ IDNO: 16.

As used herein, an “isolated nucleic acid molecule” and “isolatednucleic acid fragment” will be used interchangeably and refers to apolymer of RNA or DNA that is single- or double-stranded, optionallycontaining synthetic, non-natural or altered nucleotide bases. Anisolated nucleic acid molecule in the form of a polymer of DNA may becomprised of one or more segments of cDNA, genomic DNA or synthetic DNA.

The term “amino acid” refers to the basic chemical structural unit of aprotein or polypeptide. The following abbreviations are used herein toidentify specific amino acids:

Three-Letter One-Letter Amino Acid Abbreviation Abbreviation Alanine AlaA Arginine Arg R Asparagine Asn N Aspartic acid Asp D Cysteine Cys CGlutamine Gln Q Glutamic acid Glu E Glycine Gly G Histidine His HIsoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met MPhenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr TTryptophan Trp W Tyrosine Tyr Y Valine Val V Any amino acid or Xaa X asdefined herein

As used herein, “substantially similar” refers to nucleic acid moleculeswherein changes in one or more nucleotide bases results in the addition,substitution, or deletion of one or more amino acids, but does notaffect the functional properties (i.e. perhydrolytic activity) of theprotein encoded by the DNA sequence. As used herein, “substantiallysimilar” also refers to an enzyme having an amino acid sequence that isleast lest 40%, preferably at least 50%, more preferably at least 60%,more preferably at least 70%, even more preferably at least 80%, yeteven more preferably at least 90%, and most preferably at least 95%identical to the sequences reported herein wherein the resulting enzymeretains the present functional properties (i.e., perhydrolyticactivity). “Substantially similar” may also refer to an enzyme havingperhydrolytic activity encoded by nucleic acid molecules that hybridizesunder stringent conditions to the nucleic acid molecules reportedherein. It is therefore understood that the invention encompasses morethan the specific exemplary sequences.

For example, it is well known in the art that alterations in a genewhich result in the production of a chemically equivalent amino acid ata given site, but do not affect the functional properties of the encodedprotein are common. For the purposes of the present inventionsubstitutions are defined as exchanges within one of the following fivegroups:

-   -   1. Small aliphatic, nonpolar or slightly polar residues: Ala,        Ser, Thr (Pro, Gly);    -   2. Polar, negatively charged residues and their amides: Asp,        Asn, Glu, Gln;    -   3. Polar, positively charged residues: His, Arg, Lys;    -   4. Large aliphatic, nonpolar residues: Met, Leu, Ile, Val (Cys);        and    -   5. Large aromatic residues: Phe, Tyr, Trp.        Thus, a codon for the amino acid alanine, a hydrophobic amino        acid, may be substituted by a codon encoding another less        hydrophobic residue (such as glycine) or a more hydrophobic        residue (such as valine, leucine, or isoleucine). Similarly,        changes which result in substitution of one negatively charged        residue for another (such as aspartic acid for glutamic acid) or        one positively charged residue for another (such as lysine for        arginine) can also be expected to produce a functionally        equivalent product. In many cases, nucleotide changes which        result in alteration of the N-terminal and C-terminal portions        of the protein molecule would also not be expected to alter the        activity of the protein.

Each of the proposed modifications is well within the routine skill inthe art, as is determination of retention of biological activity of theencoded products. Moreover, the skilled artisan recognizes thatsubstantially similar sequences are encompassed by the presentinvention. In one embodiment, substantially similar sequences aredefined by their ability to hybridize, under stringent conditions(0.1×SSC, 0.1% SDS, 65° C. and washed with 2×SSC, 0.1% SDS followed by0.1×SSC, 0.1% SDS, 65° C.) with the sequences exemplified herein. In oneembodiment, the present invention includes enzymes having perhydrolaseactivity encoded by isolated nucleic acid molecules that hybridize understringent conditions to the nucleic acid molecules reported herein. In apreferred embodiment, the present invention includes an enzyme havingperhydrolase activity encoded by isolated nucleic acid molecule thathybridize under stringent conditions to a nucleic acid molecule havingan nucleic acid sequence selected from the group consisting of SEQ IDNO: 1, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ IDNO: 13, SEQ ID NO: 15, SEQ ID NO: 31, SEQ ID NO: 41, and SEQ ID NO: 60.

As used herein, a nucleic acid molecule is “hybridizable” to anothernucleic acid molecule, such as a cDNA, genomic DNA, or RNA, when asingle strand of the first molecule can anneal to the other moleculeunder appropriate conditions of temperature and solution ionic strength.Hybridization and washing conditions are well known and exemplified inSambrook, J. and Russell, D., T. Molecular Cloning: A Laboratory Manual,Third Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor(2001). The conditions of temperature and ionic strength determine the“stringency” of the hybridization. Stringency conditions can be adjustedto screen for moderately similar molecules, such as homologous sequencesfrom distantly related organisms, to highly similar molecules, such asgenes that duplicate functional enzymes from closely related organisms.Post-hybridization washes typically determine stringency conditions. Oneset of preferred conditions uses a series of washes starting with 6×SSC,0.5% SDS at room temperature for 15 min, then repeated with 2×SSC, 0.5%SDS at 45° C. for 30 min, and then repeated twice with 0.2×SSC, 0.5% SDSat 50° C. for 30 min. A more preferred set of conditions uses highertemperatures in which the washes are identical to those above except forthe temperature of the final two 30 min washes in 0.2×SSC, 0.5% SDS wasincreased to 60° C. Another preferred set of stringent hybridizationconditions is 0.1×SSC, 0.1% SDS, 65° C. and washed with 2×SSC, 0.1% SDSfollowed by a final wash of 0.1×SSC, 0.1% SDS, 65° C. with the sequencesexemplified herein.

Hybridization requires that the two nucleic acids contain complementarysequences, although depending on the stringency of the hybridization,mismatches between bases are possible. The appropriate stringency forhybridizing nucleic acids depends on the length of the nucleic acids andthe degree of complementation, variables well known in the art. Thegreater the degree of similarity or homology between two nucleotidesequences, the greater the value of Tm for hybrids of nucleic acidshaving those sequences. The relative stability (corresponding to higherTm) of nucleic acid hybridizations decreases in the following order:RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greater than 100 nucleotidesin length, equations for calculating Tm have been derived (Sambrook andRussell, supra). For hybridizations with shorter nucleic acids, i.e.,oligonucleotides, the position of mismatches becomes more important, andthe length of the oligonucleotide determines its specificity (Sambrookand Russell, supra). In one aspect, the length for a hybridizablenucleic acid is at least about 10 nucleotides. Preferably, a minimumlength for a hybridizable nucleic acid is at least about 15 nucleotidesin length, more preferably at least about 20 nucleotides in length, evenmore preferably at least 30 nucleotides in length, even more preferablyat least 300 nucleotides in length, and most preferably at least 800nucleotides in length. Furthermore, the skilled artisan will recognizethat the temperature and wash solution salt concentration may beadjusted as necessary according to factors such as length of the probe.

As used herein, the term “percent identity” is a relationship betweentwo or more polypeptide sequences or two or more polynucleotidesequences, as determined by comparing the sequences. In the art,“identity” also means the degree of sequence relatedness betweenpolypeptide or polynucleotide sequences, as the case may be, asdetermined by the match between strings of such sequences. “Identity”and “similarity” can be readily calculated by known methods, includingbut not limited to those described in: Computational Molecular Biology(Lesk, A. M., ed.) Oxford University Press, NY (1988); Biocomputing:Informatics and Genome Projects (Smith, D. W., ed.) Academic Press, NY(1993); Computer Analysis of Sequence Data, Part I (Griffin, A. M., andGriffin, H. G., eds.) Humana Press, NJ (1994); Sequence Analysis inMolecular Biology (von Heinje, G., ed.) Academic Press (1987); andSequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) StocktonPress, NY (1991). Methods to determine identity and similarity arecodified in publicly available computer programs. Sequence alignmentsand percent identity calculations may be performed using the Megalignprogram of the LASERGENE bioinformatics computing suite (DNASTAR Inc.,Madison, Wis.), the AlignX program of Vector NTI v, 7.0 (Informax, Inc.,Bethesda, Md.), or the EMBOSS Open Software Suite (EMBL-EBI; Rice etal., Trends in Genetics 16, (6) pp 276-277 (2000)). Multiple alignmentof the sequences can be performed using the Clustal method (i.e.CLUSTALW; for example version 1.83) of alignment (Higgins and Sharp,CABIOS, 5:151-153 (1989); Higgins et al., Nucleic Acids Res.22:4673-4680 (1994); and Chenna et al., Nucleic Acids Res 31(13):3497-500 (2003)), available from the European Molecular BiologyLaboratory via the European Bioinformatics Institute) with the defaultparameters. Suitable parameters for CLUSTALW protein alignments includeGAP Existence penalty=15, GAP extension=0.2, matrix=Gonnet (e.g.Gonnet250), protein ENDGAP=−1, Protein GAPDIST=4, and KTUPLE=1. In oneembodiment, a fast or slow alignment is used with the default settingswhere a slow alignment is preferred. Alternatively, the parameters usingthe CLUSTALW method (version 1.83) may be modified to also use KTUPLE=1,GAP PENALTY=10, GAP extension=1, matrix=BLOSUM (e.g. BLOSUM64),WINDOW=5, and TOP DIAGONALS SAVED=5.

In one aspect of the present invention, suitable isolated nucleic acidmolecules (isolated polynucleotides of the present invention) encode apolypeptide having an amino acid sequence that is at least about 50%,preferably at least 60%, more preferably at least 70%, more preferablyat least 80%, even more preferably at least 85%, even more preferably atleast 90%, and most preferably at least 95% identical to the amino acidsequences reported herein. Suitable nucleic acid molecules of thepresent invention not only have the above homologies, but also typicallyencode a polypeptide having about 300 to about 340 amino acids, morepreferably about 310 to about 330 amino acids, and most preferably about318 amino acids.

As used herein, the terms “signature motif”, “CE-7 signature motif”, and“diagnostic motif” refer to conserved structures shared among a familyof enzymes having a defined activity. The signature motif can be used todefine and/or identify the family of structurally related enzymes havingsimilar enzymatic activity for a defined family of substrates. Thesignature motif can be a single contiguous amino acid sequence or acollection of discontiguous, conserved motifs that together form thesignature motif. Typically, the conserved motif(s) is represented by anamino acid sequence. As described herein, the present perhydrolasesbelong to the family of CE-7 carbohydrate esterases. This family ofenzymes can be defined by the presence of a signature motif (Vincent etal., supra). As defined herein, the signature motif for CE-7 esterasescomprises 3 conserved motifs (residue position numbering relative toreference sequence SEQ ID NO: 2):

a) Arg118-Gly119-Gln120;

b) Gly179-Xaa180-Ser181-Gln182-Gly183; and

c) His298-Glu299.

The Xaa at amino acid residue position 180 is typically glycine oralanine. Amino acid residues belonging to the catalytic triad are inbold.

Further analysis of the conserved motifs within the CE-7 carbohydrateesterase family indicates the presence of an additional conserved motif(LXD at amino acid positions 267-269 of SEQ ID NO: 2) that may be tofurther define a perhydrolase belonging to the CE-7 carbohydrateesterase family (FIGS. 1 a-c). In a further embodiment, the signaturemotif defined above includes a forth conserved motif defined as:

Leu267-Xaa268-Asp269;

The Xaa at amino acid residue position 268 is typically isoleucine,valine, or methionine. The forth motif includes the aspartic acidresidue (bold) belonging to the catalytic triad (Ser181-Asp269-His298).

A number of well-known global alignment algorithms (i.e. sequenceanalysis software) may be used to align two or more amino acid sequencesrepresenting enzymes having perhydrolase activity to determine is theenzyme is comprised of the present signature motif. The alignedsequence(s) are compared to the reference sequence (SEQ ID NO: 2) todetermine the existence of the signature motif. In one embodiment, aCLUSTAL alignment (CLUSTALW) using a reference amino acid sequence (asused herein the perhydrolase sequence (SEQ ID NO: 2) from the Bacillussubtilis ATCC 31954™) is used to identify perhydrolases belonging to theCE-7 esterase family. The relative numbering of the conserved amino acidresidues is based on the residue numbering of the reference amino acidsequence to account for small insertions or deletions (for example, 5amino acids of less) within the aligned sequence.

Examples of other suitable algorithms that may be used to identifysequences comprising the present signature motif (when compared to thereference sequence) include, but are not limited to Needleman and Wunsch(J. Mol. Biol. 48, 443-453 (1970); a global alignment tool) andSmith-Waterman (J. Mol. Biol. 147:195-197 (1981); a local alignmenttool). In one embodiment, a Smith-Waterman alignment is implementedusing default parameters. An example of suitable default parametersinclude the use of a BLOSUM62 scoring matrix with GAP open penalty 10and a GAP extension penalty=0.5.

A comparison of the overall percent identity among perhydrolasesexemplified herein indicates that enzymes having as little as 42%identity to SEQ ID NO: 2 (while retaining the signature motif) exhibitsignificant perhydrolase activity and are structurally classified asCE-7 carbohydrate esterases. In one embodiment, the presentperhydrolases include enzymes comprising the present signature motif andat least 40%, preferably at least 42%, more preferably at least 50%,even more preferably at least 60%, yet even more preferably at least70%, even more preferably at least 80%, yet even more preferably atleast 90%, and most preferably at least 95% identity to SEQ ID NO: 2.

Alternatively, a contiguous amino acid sequence comprising the regionencompassing the conserved motifs (i.e. amino acid residues 118-299 ofSEQ ID NO: 2; provided separately as SEQ ID NO: 63) may also be used toidentify CE-7 carbohydrate esterase family members. In anotherembodiment, suitable perhydrolases are those having perhydrolaseactivity and at least 40%, preferably at least 50%, more preferably atleast 60%, more preferably at least 70%, more preferably 80%, even morepreferably 90%, and most preferably at least 95% amino acid identity toSEQ ID NO: 61.

As used herein, “codon degeneracy” refers to the nature of the geneticcode permitting variation of the nucleotide sequence without affectingthe amino acid sequence of an encoded polypeptide. Accordingly, thepresent invention relates to any nucleic acid molecule that encodes allor a substantial portion of the amino acid sequences encoding thepresent microbial polypeptide. The skilled artisan is well aware of the“codon-bias” exhibited by a specific host cell in usage of nucleotidecodons to specify a given amino acid. Therefore, when synthesizing agene for improved expression in a host cell, it is desirable to designthe gene such that its frequency of codon usage approaches the frequencyof preferred codon usage of the host cell.

As used herein, “synthetic genes” can be assembled from oligonucleotidebuilding blocks that are chemically synthesized using procedures knownto those skilled in the art. These building blocks are ligated andannealed to form gene segments that are then enzymatically assembled toconstruct the entire gene. “Chemically synthesized”, as pertaining to aDNA sequence, means that the component nucleotides were assembled invitro. Manual chemical synthesis of DNA may be accomplished usingwell-established procedures, or automated chemical synthesis can beperformed using one of a number of commercially available machines.Accordingly, the genes can be tailored for optimal gene expression basedon optimization of nucleotide sequences to reflect the codon bias of thehost cell. The skilled artisan appreciates the likelihood of successfulgene expression if codon usage is biased towards those codons favored bythe host. Determination of preferred codons can be based on a survey ofgenes derived from the host cell where sequence information isavailable.

As used herein, “gene” refers to a nucleic acid molecule that expressesa specific protein, including regulatory sequences preceding (5′non-coding sequences) and following (3′ non-coding sequences) the codingsequence. “Native gene” refers to a gene as found in nature with its ownregulatory sequences. “Chimeric gene” refers to any gene that is not anative gene, comprising regulatory and coding sequences that are notfound together in nature. Accordingly, a chimeric gene may compriseregulatory sequences and coding sequences that are derived fromdifferent sources, or regulatory sequences and coding sequences derivedfrom the same source, but arranged in a manner different from that foundin nature. “Endogenous gene” refers to a native gene in its naturallocation in the genome of an organism. A “foreign” gene refers to a genenot normally found in the host organism, but that is introduced into thehost organism by gene transfer. Foreign genes can comprise native genesinserted into a non-native organism, or chimeric genes. A “transgene” isa gene that has been introduced into the genome by a transformationprocedure.

As used herein, “coding sequence” refers to a DNA sequence that codesfor a specific amino acid sequence. “Suitable regulatory sequences”refer to nucleotide sequences located upstream (5′ non-codingsequences), within, or downstream (3′ non-coding sequences) of a codingsequence, and which influence the transcription, RNA processing orstability, or translation of the associated coding sequence. Regulatorysequences may include promoters, translation leader sequences, RNAprocessing site, effector binding site and stern-loop structure.

As used herein, “promoter” refers to a DNA sequence capable ofcontrolling the expression of a coding sequence or functional RNA. Ingeneral, a coding sequence is located 3′ to a promoter sequence.Promoters may be derived in their entirety from a native gene, or becomposed of different elements derived from different promoters found innature, or even comprise synthetic DNA segments. It is understood bythose skilled in the art that different promoters may direct theexpression of a gene at different stages of development, or in responseto different environmental or physiological conditions. Promoters thatcause a gene to be expressed at most times are commonly referred to as“constitutive promoters”. It is further recognized that since in mostcases the exact boundaries of regulatory sequences have not beencompletely defined, DNA fragments of different lengths may haveidentical promoter activity.

As used herein, the “3′ non-coding sequences” refer to DNA sequenceslocated downstream of a coding sequence and include polyadenylationrecognition sequences (normally limited to eukaryotes) and othersequences encoding regulatory signals capable of affecting mRNAprocessing or gene expression. The polyadenylation signal is usuallycharacterized by affecting the addition of polyadenylic acid tracts(normally limited to eukaryotes) to the 3′ end of the mRNA precursor.

As used herein, the term “operably linked” refers to the association ofnucleic acid sequences on a single nucleic acid molecule so that thefunction of one is affected by the other. For example, a promoter isoperably linked with a coding sequence when it is capable of affectingthe expression of that coding sequence, i.e., that the coding sequenceis under the transcriptional control of the promoter. Coding sequencescan be operably linked to regulatory sequences in sense or antisenseorientation.

As used herein, the term “expression” refers to the transcription andstable accumulation of sense (mRNA) or antisense RNA derived from thenucleic acid molecule of the invention. Expression may also refer totranslation of mRNA into a polypeptide.

As used herein, “transformation” refers to the transfer of a nucleicacid molecule into the genome of a host organism, resulting ingenetically stable inheritance. In the present invention, the hostcell's genome includes chromosomal and extrachromosomal (e.g. plasmid)genes. Host organisms containing the transformed nucleic acid moleculesare referred to as “transgenic” or “recombinant” or “transformed”organisms.

As used herein, the terms “plasmid”, “vector” and “cassette” refer to anextrachromosomal element often carrying genes which are not part of thecentral metabolism of the cell, and usually in the form of circulardouble-stranded DNA molecules. Such elements may be autonomouslyreplicating sequences, genome integrating sequences, phage or nucleotidesequences, linear or circular, of a single- or double-stranded DNA orRNA, derived from any source, in which a number of nucleotide sequenceshave been joined or recombined into a unique construction which iscapable of introducing a promoter fragment and DNA sequence for aselected gene product along with appropriate 3′ untranslated sequenceinto a cell. “Transformation cassette” refers to a specific vectorcontaining a foreign gene and having elements in addition to the foreigngene that facilitate transformation of a particular host cell.“Expression cassette” refers to a specific vector containing a foreigngene and having elements in addition to the foreign gene that allow forenhanced expression of that gene in a foreign host.

As used herein, the term “sequence analysis software” refers to anycomputer algorithm or software program that is useful for the analysisof nucleotide or amino acid sequences. “Sequence analysis software” maybe commercially available or independently developed. Typical sequenceanalysis software will include, but is not limited to, the GCG suite ofprograms (Wisconsin Package Version 9.0, Genetics Computer Group (GCG),Madison, Wis.), BLASTP, BLASTN, BLASTX (Altschul et al., J. Mol. Biol.215:403-410 (1990), and DNASTAR (DNASTAR, Inc. 1228 S. Park St. Madison,Wis. 53715 USA), CLUSTALW (for example, version 1.83; Thompson et al.,Nucleic Acids Research, 22(22):4673-4680 (1994), and the FASTA programincorporating the Smith-Waterman algorithm (W. R. Pearson, Comput.Methods Genome Res., [Proc. Int. Symp.] (1994), Meeting Date 1992,111-20. Editor(s): Suhai, Sandor. Publisher: Plenum, New York, N.Y.),Vector NTI (Informax, Bethesda, Md.) and Sequencher v. 4.05. Within thecontext of this application it will be understood that where sequenceanalysis software is used for analysis, that the results of the analysiswill be based on the “default values” of the program referenced, unlessotherwise specified. As used herein “default values” will mean any setof values or parameters set by the software manufacturer that originallyload with the software when first initialized.

As used herein, the term “biological contaminants” refers to one or moreunwanted and/or pathogenic biological entities including, but notlimited to microorganisms, spores, viruses, prions, and mixturesthereof. The process produces an efficacious concentration of at leastone percarboxylic acid useful to reduce and/or eliminate the presence ofthe viable biological contaminants. In a preferred embodiment, themicrobial contaminant is a viable pathogenic microorganism.

As used herein, the term “disinfect” refers to the process ofdestruction of or prevention of the growth of biological contaminants.As used herein, the term “disinfectant” refers to an agent thatdisinfects by destroying, neutralizing, or inhibiting the growth ofbiological contaminants. Typically, disinfectants are used to treatinanimate objects or surfaces. As used herein, the term “antiseptic”refers to a chemical agent that inhibits the growth of disease-carryingmicroorganisms. In one aspect of the embodiment, the biologicalcontaminants are pathogenic microorganisms.

As used herein, the term “virucide” refers to an agent that inhibits ordestroys viruses, and is synonymous with “viricide”. An agent thatexhibits the ability to inhibit or destroy viruses is described ashaving “virucidal” activity. Peracids can have virucidal activity.Typical alternative virucides known in the art which may be suitable foruse with the present invention include, for example, alcohols, ethers,chloroform, formaldehyde, phenols, beta propiolactone, iodine, chlorine,mercury salts, hydroxylamine, ethylene oxide, ethylene glycol,quaternary ammonium compounds, enzymes, and detergents.

As used herein, the term “biocide” refers to a chemical agent, typicallybroad spectrum, which inactivates or destroys microorganisms. A chemicalagent that exhibits the ability to inactivate or destroy microorganismsis described as having “biocidal” activity. Peracids can have biocidalactivity. Typical alternative biocides known in the art, which may besuitable for use in the present invention include, for example,chlorine, chlorine dioxide, chloroisocyanurates, hypochlorites, ozone,acrolein, amines, chlorinated phenolics, copper salts, organo-sulphurcompounds, and quaternary ammonium salts.

As used herein, the phrase “minimum biocidal concentration” refers tothe minimum concentration of a biocidal agent that, for a specificcontact time, will produce a desired lethal, irreversible reduction inthe viable population of the targeted microorganisms. The effectivenesscan be measured by the log₁₀ reduction in viable microorganisms aftertreatment. In one aspect, the targeted reduction in viablemicroorganisms after treatment is at least a 3-log reduction, morepreferably at least a 4-log reduction, and most preferably at least a5-log reduction. In another aspect, the minimum biocidal concentrationis at least a 6-log reduction in viable microbial cells.

As used herein, the terms “peroxygen source” and “source of peroxygen”refer to compounds capable of providing hydrogen peroxide at aconcentration of about 1 mM or more when in an aqueous solutionincluding, but not limited to hydrogen peroxide, hydrogen peroxideadducts (e.g., urea-hydrogen peroxide adduct (carbamide peroxide)),perborates, and percarbonates. As described herein, the concentration ofthe hydrogen peroxide provided by the peroxygen compound in the aqueousreaction mixture is initially at least 1 mM or more upon combining thereaction components. In one embodiment, the hydrogen peroxideconcentration in the aqueous reaction mixture is at least 10 mM. Inanother embodiment, the hydrogen peroxide concentration in the aqueousreaction mixture is at least 100 mM. In another embodiment, the hydrogenperoxide concentration in the aqueous reaction mixture is at least 200mM. In another embodiment, the hydrogen peroxide concentration in theaqueous reaction mixture is 500 mM or more. In yet another embodiment,the hydrogen peroxide concentration in the aqueous reaction mixture is1000 mM or more. The molar ratio of the hydrogen peroxide to enzymesubstrate, e.g. triglyceride, (H₂O₂:substrate) in the aqueous reactionmixture may be from about 0.002 to 20, preferably about 0.1 to 10, andmost preferably about 0.5 to 5.

Suitable Reaction Conditions for the Enzyme-Catalyzed Preparation ofPeracids from Carboxylic Acid Esters and Hydrogen Peroxide

In one aspect of the invention, a process is provided to produce anaqueous mixture comprising a peracid by reacting carboxylic acid estersand an inorganic peroxide, not limited to hydrogen peroxide, sodiumperborate or sodium percarbonate, in the presence of an enzyme catalysthaving perhydrolysis activity. In one embodiment, the enzyme catalystcomprises a perhydrolase having a structure belonging to the CE-7carbohydrate esterase family. In another embodiment, the perhydrolasecatalyst is structurally classified as a cephalosporin C deacetylase. Inanother embodiment, the perhydrolase catalyst is structurally classifiedas an acetyl xylan esterase.

In one embodiment, the perhydrolase catalyst comprises an enzyme havingperhydrolysis activity and a signature motif comprising:

-   -   a) an RGQ motif as amino acid residues 118-120;    -   b) a GXSQG motif at amino acid residues 179-183; and    -   c) an HE motif as amino acid residues 298-299 when aligned to        reference sequence SEQ ID NO: 2 using CLUSTALW.

In a further embodiment, the signature motif additional comprises aforth conserved motif defined as an LXD motif at amino acid residues267-269 when aligned to reference sequence SEQ ID NO: 2 using CLUSTALW.

In another embodiment, the perhydrolase catalyst comprises an enzymehaving the present signature motif and at least 40% amino acid to SEQ IDNO: 2.

In another embodiment, the perhydrolase catalyst comprises an enzymehaving perhydrolase activity selected from the group consisting of SEQID NO: 2, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQID NO: 14, SEQ ID NO: 16, and SEQ ID NO: 32.

In another embodiment, the perhydrolase catalyst comprises an enzymehaving at least 40% amino acid identity to a contiguous signature motifdefined as SEQ ID NO: 61 wherein the conserved motifs described above(e.g. RGQ, GXSQG, and HE, and optionally, LXD) are conserved.

In another embodiment, the perhydrolase catalyst comprises an enzymehaving an amino acid sequence encoded by a nucleic acid molecule thathybridizes to a nucleic acid sequence selected from the group consistingof SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO:11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 31, SEQ ID NO: 41, and SEQID NO: 60 under stringent hybridization conditions.

In another embodiment, the perhydrolase catalyst comprises an enzymehaving an amino acid sequence selected from the group consisting of SEQID NO: 2, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQID NO: 14, SEQ ID NO: 16, and SE ID NO: 32 wherein said enzyme may haveone or more additions, deletions, or substitutions so long as thesignature motif is conserved and perhydrolase activity is retained.

Suitable carboxylic acid esters have a formula selected from the groupconsisting of:

a) esters of the formula

wherein R₁═C1 to C7 straight chain or branched chain alkyl optionallysubstituted with an hydroxyl or a C1 to C4 alkoxy group and R₂=C1 to C10straight chain or branched chain alkyl, alkenyl, alkynyl, aryl,alkylaryl, alkylheteroaryl, heteroaryl, (CH₂CH₂—O)_(n)H or(CH₂CH(CH₃)—O)_(n)H and n=1 to 10;

b) glycerides of the formula

wherein R₁═C1 to C7 straight chain or branched chain alkyl optionallysubstituted with an hydroxyl or a C1 to C4 alkoxy group and R₃ and R₄are individually H or R₁C(O); and

c) acetylated saccharides selected from the group consisting ofacetylated mono-, di-, and polysaccharides.

In a preferred embodiment, the acetylated saccharides include acetylatedmono-, di-, and polysaccharides. In another embodiment, the acetylatedsaccharides are selected from the group consisting of acetylated xylan,fragments of acetylated xylan, acetylated xylose (such as xylosetetraacetate), acetylated glucose (such as glucose pentaacetate),β-D-ribofuranose-1,2,3,5-tetraacetate, tri-O-acetyl-D-galactal, andtri-O-acetyl-D-glucal, and acetylated cellulose. In a preferredembodiment, the acetylated saccharide is selected from the groupconsisting of β-D-ribofuranose-1,2,3,5-tetraacetate,tri-O-acetyl-D-galactal, and tri-O-acetyl-D-glucal, and acetylatedcellulose. As such, acetylated carbohydrates may be suitable substratesfor generating percarboxylic acids using the present process (i.e., inthe presence of a peroxygen source).

In another aspect, the carboxylic acid ester is selected from the groupconsisting of ethyl acetate, methyl lactate, ethyl lactate, methylglycolate, ethyl glycolate, methyl methoxyacetate, ethyl methoxyacetate,methyl 3-hydroxybutyrate, ethyl 3-hydroxybutyrate, triethyl 2-acetylcitrate, glucose pentaacetate, gluconolactone, glycerides (mono-, di-,and triglycerides) such as monoacetin, diacetin, triacetin,monopropionin, dipropionin (glyceryl dipropionate), tripropionin(1,2,3-tripropionylglycerol), monobutyrin, dibutyrin (glyceryldibutyrate), tributyrin (1,2,3-tributyrylglycerol), acetylatedsaccharides, and mixtures thereof. In another aspect, the carboxylicacid ester substrates are selected from the group consisting ofmonoacetin, diacetin, triacetin, monopropionin, dipropionin,tripropionin, monobutyrin, dibutyrin, tributyrin, ethyl acetate, andethyl lactate. In yet another aspect, the carboxylic acid estersubstrates are selected from the group consisting of diacetin,triacetin, ethyl acetate, and ethyl lactate. In a preferred aspect, thecarboxylic acid ester is a glyceride selected from the group consistingof monoacetin, diacetin, triacetin, and mixtures thereof.

The carboxylic acid ester is present in the reaction mixture at aconcentration sufficient to produce the desired concentration of peracidupon enzyme-catalyzed perhydrolysis. The carboxylic acid ester need notbe completely soluble in the reaction mixture, but has sufficientsolubility to permit conversion of the ester by the perhydrolasecatalyst to the corresponding peracid. The carboxylic acid ester ispresent in the reaction mixture at a concentration of 0.05 wt % to 40 wt% of the reaction mixture, preferably at a concentration of 0.1 wt % to20 wt % of the reaction mixture, and more preferably at a concentrationof 0.5 wt % to 10 wt % of the reaction mixture.

The peroxygen source may include, but is not limited to, hydrogenperoxide, hydrogen peroxide adducts (e.g., urea-hydrogen peroxide adduct(carbamide peroxide)) perborate salts and percarbonate salts. Theconcentration of peroxygen compound in the reaction mixture may rangefrom 0.0033 wt % to about 50 wt %, preferably from 0.033 wt % to about40 wt %, more preferably from 0.33 wt % to about 30 wt %.

Many perhydrolase catalysts (whole cells, permeabilized whole cells, andpartially purified whole cell extracts) have been reported to havecatalase activity (EC 1.11.1.6). Catalases catalyze the conversion ofhydrogen peroxide into oxygen and water. In one aspect, theperhydrolysis catalyst lacks catalase activity. In another aspect, acatalase inhibitor is added to the reaction mixture. Examples ofcatalase inhibitors include, but are not limited to, sodium azide andhydroxylamine sulfate. One of skill in the art can adjust theconcentration of catalase inhibitor as needed. The concentration of thecatalase inhibitor typically ranges from 0.1 mM to about 1 M; preferablyabout 1 mM to about 50 mM; more preferably from about 1 mM to about 20mM. In one aspect, sodium azide concentration typically ranges fromabout 20 mM to about 60 mM while hydroxylamine sulfate is concentrationis typically about 0.5 mM to about 30 mM, preferably about 10 mM.

In another embodiment, the enzyme catalyst lacks significant catalaseactivity or is engineered to decrease or eliminate catalase activity.The catalase activity in a host cell can be down-regulated or eliminatedby disrupting expression of the gene(s) responsible for the catalaseactivity using well known techniques including, but not limited to,transposon mutagenesis, RNA antisense expression, targeted mutagenesis,and random mutagenesis. In a preferred embodiment, the gene(s) encodingthe endogenous catalase activity are down-regulated or disrupted (i.e.knocked-out). As used herein, a “disrupted” gene is one where theactivity and/or function of the protein encoded by the modified gene isno longer present. Means to disrupt a gene are well-known in the art andmay include, but are not limited to insertions, deletions, or mutationsto the gene so long as the activity and/or function of the correspondingprotein is no longer present. In a further preferred embodiment, theproduction host is an E. coli production host comprising a disruptedcatalase gene selected from the group consisting of katG (SEQ ID NO: 47)and katE (SEQ ID NO: 56). In another embodiment, the production host isan E. coli strain comprising a down-regulation and/or disruption in bothkatg1 and a katE catalase genes. An E. coli strain comprising adouble-knockout of katG and katE is provided herein (see Example 15; E.coli strain KLP18).

The catalase negative E. coli strain KLP18 (katG and katE doubleknockout) that was constructed (Example 15) was demonstrated to be asuperior host for large scale (10-L and greater) production ofperhydrolase enzymes compared to the catalase negative strain UM2 (E.coli Genetic Stock Center #7156, Yale University, New Haven Conn.), asdetermined by growth under fermenter conditions (Examples 17-19).Although both KLP18 and UM2 are catalase-negative strains, UM2 is knownto have numerous nutritional auxotrophies, and therefore requires mediathat is enriched with yeast extract and peptone. Even when employingenriched media for fermentation, UM2 grew poorly and to a limitedmaximum cell density (OD). In contrast, KLP18 had no special nutritionalrequirements and grew to high cell densities on mineral media alone orwith additional yeast extract (Example 20).

The concentration of the catalyst in the aqueous reaction mixturedepends on the specific catalytic activity of the catalyst, and ischosen to obtain the desired rate of reaction. The weight of catalyst inperhydrolysis reactions typically ranges from 0.0005 mg to 10 mg per mLof total reaction volume, preferably from 0.010 mg to 2.0 mg per mL. Thecatalyst may also be immobilized on a soluble or insoluble support usingmethods well-known to those skilled in the art; see for example,Immobilization of Enzymes and Cells; Gordon F. Bickerstaff, Editor;Humana Press, Totowa, N.J., USA; 1997. The use of immobilized catalystspermits the recovery and reuse of the catalyst in subsequent reactions.The enzyme catalyst may be in the form of whole microbial cells,permeabilized microbial cells, microbial cell extracts,partially-purified or purified enzymes, and mixtures thereof.

In one aspect, the concentration of peracid generated by the combinationof chemical perhydrolysis and enzymatic perhydrolysis of the carboxylicacid ester is sufficient to provide an effective concentration ofperacid for bleaching or disinfection at a desired pH. In anotheraspect, the present methods provide combinations of enzymes and enzymesubstrates to produce the desired effective concentration of peracid,where, in the absence of added enzyme, there is a significantly lowerconcentration of peracid produced. Although there may in some cases besubstantial chemical perhydrolysis of the enzyme substrate by directchemical reaction of inorganic peroxide with the enzyme substrate, theremay not be a sufficient concentration of peracid generated to provide aneffective concentration of peracid in the desired applications, and asignificant increase in total peracid concentration is achieved by theaddition of an appropriate perhydrolase catalyst to the reactionmixture.

The concentration of peracid generated (e.g. peracetic acid) by theperhydrolysis of at least one carboxylic acid ester is at least about 20ppm, preferably at least 100 ppm, more preferably at least about 200 ppmperacid, more preferably at least 300 ppm, more preferably at least 500ppm, more preferably at least 700 ppm, more preferably at least about1000 ppm peracid, most preferably at least 2000 ppm peracid within 10minutes, preferably within 5 minutes, of initiating the perhydrolysisreaction. The product mixture comprising the peracid may be optionallydiluted with water, or a solution predominantly comprised of water, toproduce a mixture with the desired lower concentration of peracid. Inone aspect, the reaction time required to produce the desiredconcentration of peracid is not greater than about two hours, preferablynot greater than about 30 minutes, more preferably not greater thanabout 10 minutes, and most preferably in about 5 minutes or less. Inother aspects, a hard surface or inanimate object contaminated with aconcentration of a microbial population is contacted with the peracidformed in accordance with the processes described herein within about 5minutes to about 168 hours of combining said reaction components, orwithin about 5 minutes to about 48 hours, or within about 5 minutes to 2hours of combining said reaction components, or any such time intervaltherein.

The temperature of the reaction is chosen to control both the reactionrate and the stability of the enzyme catalyst activity. The temperatureof the reaction may range from just above the freezing point of thereaction mixture (approximately 0° C.) to about 75° C., with a preferredrange of reaction temperature of from about 5° C. to about 55° C.

The pH of the final reaction mixture containing peracid is from about 2to about 9, preferably from about 3 to about 8, more preferably fromabout 5 to about 8, even more preferably about 6 to about 8, and yeteven more preferably about 6.5 to about 7.5. In another embodiment, thepH of the reaction mixture is acidic (pH<7). The pH of the reaction, andof the final reaction mixture, may optionally be controlled by theaddition of a suitable buffer, including, but not limited to phosphate,pyrophosphate, bicarbonate, acetate, or citrate. The concentration ofbuffer, when employed, is typically from 0.1 mM to 1.0 M, preferablyfrom 1 mM to 300 mM, most preferably from 10 mM to 100 mM.

In another aspect, the enzymatic perhydrolysis product may containadditional components that provide desirable functionality. Theseadditional components include, but are not limited to buffers, detergentbuilders, thickening agents, emulsifiers, surfactants, wetting agents,corrosion inhibitors (e.g., benzotriazole), enzyme stabilizers, andperoxide stabilizers (e.g., metal ion chelating agents). Many of theadditional components are well known in the detergent industry (see forexample U.S. Pat. No. 5,932,532; hereby incorporated by reference).Examples of emulsifiers include, but are not limited to polyvinylalcohol or polyvinylpyrrolidone. Examples of thickening agents include,but are not limited to LAPONITE® RD, corn starch, PVP, CARBOWAX®,CARBOPOL®, CABOSIL®, polysorbate 20, PVA, and lecithin. Examples ofbuffering systems include, but are not limited to sodium phosphatemonobasic/sodium phosphate dibasic; sulfamic acid/triethanolamine;citric acid/triethanolamine; tartaric acid/triethanolamine; succinicacid/triethanolamine; and acetic acid/triethanolamine. Examples ofsurfactants include, but are not limited to a) non-ionic surfactantssuch as block copolymers of ethylene oxide or propylene oxide,ethoxylated or propoxylated linear and branched primary and secondaryalcohols, and aliphatic phosphine oxides b) cationic surfactants such asquaternary ammonium compounds, particularly quaternary ammoniumcompounds having a C8-C20 alkyl group bound to a nitrogen atomadditionally bound to three C1-C2 alkyl groups, c) anionic surfactantssuch as alkane carboxylic acids (e.g., C8-C20 fatty acids), alkylphosphonates, alkane sulfonates (e.g., sodium dodecylsulphate “SDS”) orlinear or branched alkyl benzene sulfonates, alkene sulfonates and d)amphoteric and zwitterionic surfactants such as aminocarboxylic acids,aminodicarboxylic acids, alkybetaines, and mixtures thereof. Additionalcomponents may include fragrances, dyes, stabilizers of hydrogenperoxide (e.g., metal chelators such as1-hydroxyethylidene-1,1-diphosphonic acid (DEQUEST® 2010, Solutia Inc.,St. Louis, Mo. and ethylenediaminetetraacetic acid (EDTA)), TURPINAL®SL, DEQUEST® 0520, DEQUEST® 0531, stabilizers of enzyme activity (e.g.,polyethyleneglycol (PEG)), and detergent builders.

In Situ Production of Peracids using a Perhydrolase Catalyst

Cephalosporin C deacetylases (E.C. 3.1.1.41; systematic namecephalosporin C acetylhydrolases; CAHs) are enzymes having the abilityto hydrolyze the acetyl ester bond on cephalosporins such ascephalosporin C, 7-aminocephalosporanic acid, and7-(thiophene-2-acetamido)cephalosporanic acid (Abbott, B. and Fukuda,D., Appl. Microbiol. 30(3):413-419 (1975)). CAHs belong to a largerfamily of structurally related enzymes referred to as the carbohydrateesterase family seven (CE-7; see Coutinho, P. M., Henrissat, B.“Carbohydrate-active enzymes: an integrated database approach” in RecentAdvances in Carbohydrate Bioengineering, H. J. Gilbert, G. Davies, B.Henrissat and B. Svensson eds., (1999) The Royal Society of Chemistry,Cambridge, pp. 3-12.)

The CE-7 family includes both CAHs and acetyl xylan esterases (AXEs;E.C. 3.1.1.72). CE-7 family members share a common structural motif andare quite unusual in that they typically exhibit ester hydrolysisactivity for both acetylated xylooliogsaccharides and cephalosporin C,suggesting that the CE-7 family represents a single class of proteinswith a multifunctional deacetylase activity against a range of smallsubstrates (Vincent et al., J. Mol. Biol., 330:593-606 (2003)). Vincentet al. describes the structural similarity among the members of thisfamily and defines a signature sequence motif characteristic of the CE-7family.

Members of the CE-7 family are found in plants, fungi (e.g.,Cephalosporidium acremonium), yeasts (e.g., Rhodosporidium toruloides,Rhodotorula glutinis), and bacteria such as Thermoanaerobacterium sp.;Norcardia lactamdurans, and various members of the genus Bacillus(Politino et al., Appl. Environ. Microbiol., 63(12):4807-4811 (1997);Sakai et al., J. Ferment. Bioeng. 85:53-57 (1998); Lorenz, W. andWiegel, J., J. Bacteria 179:5436-5441 (1997); Cardoza et al., Appl.Microbiol. Biotechnol., 54(3):406-412 (2000); Mitshushima et al., supra,Abbott, B. and Fukuda, D., Appl. Microbiol. 30(3):413-419 (1975);Vincent et al., supra, Takami et al., NAR, 28(21):4317-4331 (2000); Reyet al., Genome Biol., 5(10): article 77 (2004); Degrassi et al.,Microbiology., 146:1585-1591 (2000); U.S. Pat. No. 6,645,233; U.S. Pat.No. 5,281,525; U.S. Pat. No. 5,338,676; and WO 99/03984. Anon-comprehensive list of CE-7 carbohydrate esterase family membershaving significant homology to SEQ ID NO: 2 are provided in Table 1.

TABLE 1 Example of CE-7 Enzymes Having Significant Homology to SEQ IDNO: 2. % Amino Source Organism Acid (GENBANK ® Nucleotide Amino AcidIdentity to Accession No. of Sequence Sequence SEQ ID the CE-7 enzyme)(SEQ ID NO:) (SEQ ID NO:) NO: 2. Reference B. subtilis 1 2 100 B.subtilis ATCC 31954 ™ SHS 0133 Mitshushima et al. supra B. subtilissubsp. 5 6 98 Kunst et al., subtilis str. 168 supra. (NP_388200)WO99/03984 B. subtilis BE1010 Payne and Jackson, J. Bacteriol. 173:2278-2282 (1991)) B. subtilis 7 8 96 U.S. Pat. No. 6,465,233 ATCC 6633(YP_077621.1) B. licheniformis 9 10 77 Rey et al., supra ATCC 14580(YP_077621.1) B. pumilus PS213 11, 60 12 76 Degrassi et al.,(CAB76451.2) supra Clostridium 13 14 57 Copeland et al. thermocellum USDept. of ATCC 27405 Energy Joint (ZP_00504991) Genome Institute (JGI-PGF) Direct Submission GENBANK ® ZP_00504991 Thermotoga 15, 41 16 42 Seeneapolitana GENBANK ® (AAB70869.1) AAB70869.1 Thermotoga 17 18 42 Nelsonet al., maritima MSB8 Nature 399 (NP_227893.1) (6734): 323-329 (1999)Bacillus sp. 21 22 40 Siefert at al. NRRL B-14911 J. Craig Venter(ZP_01168674) Institute. Direct Submission Under GENBANK ® ZP_01168674Thermoanaerobac 19 20 37 Lorenz and terium sp. Wiegel, supra(AAB68821.1) Bacillus 23 24 36 Takami et al., halodurans C-125 supra(NP_244192) Bacillus clausii 25 26 33 Kobayashi et KSM-K16 al., Appl.(YP_175265) Microbiol. Biotechnol. 43 (3), 473-481 (1995)

The present perhydrolases are all members of the CE-7 carbohydrateesterase family. As described by Vincent et al. (supra), members of thefamily share a common signature motif that is characteristic of thisfamily. A CLUSTALW alignment of the present perhydrolases illustratesthat all of the members belong to the CE-7 carbohydrate esterase family(FIGS. 1 a-c). A comparison of the overall percent amino acid identityamount the present perhydrolases is provided in Table 2.

TABLE 2 Percent Amino Acid Identity Between Perhydrolases¹ B. subtilisB. subtilis B. subtilis ATCC B. subtilis ATCC ATCC B. Licheniformis B.pumilus C. Thermocellum Thermotoga 31954 BE1010 29233 6633 ATCC 14580PS213 ATCC 27405 Neapolitana B. subtilis 100 98 99 96 77 76 57 42 ATCC31954 (SEQ ID NO: 2) B. subtilis 98 100 99 96 76 77 57 43 BE1010 (SEQ IDNO: 6) B. subtilis 99 99 100 96 77 76 57 43 ATCC 29233 (SEQ ID NO: 34)B. subtilis ATCC 96 96 96 100 76 76 56 43 6633 (SEQ ID NO: 8) B.licheniformis 77 76 77 76 100 69 56 45 ATCC 14580 (SEQ ID NO: 10) B.pumilus 76 77 76 76 69 100 57 42 PS213 (SEQ ID NO: 12) Clostridium 57 5757 56 56 57 100 45 Thermocellum ATCC 27405 (SEQ ID NO: 14) Thermotoga 4243 43 43 45 42 45 100 neapolitana (SEQ ID NO: 16) ¹Percent identitydetermined using blast2seq algorithm using BLOSUM62, gap open = 11, gapextension = 1, x_drop = 0, expect = 10, and wordsize = 3. Tatlana A.Tatusova, Thomas L. Madden (1999), “Blast 2 sequences - a new tool forcomparing protein and nucleotide sequences”, FEMS Microbiol Lett. 174:247-250

Although variation is observed in terms of overall percent amino acididentity (i.e. the Clostridium thermocellum ATCC 27405™ perhydrolase;SEQ ID NO: 14 shares only 57% amino acid identity with the Bacillussubtilis ATCC 31954™ perhydrolase; SEQ ID NO: 2 while the Thermotoganeapolitana perhydrolase (SEQ ID NO: 16) shares only 42% identity withSEQ ID NO: 2), each of the present perhydrolase enzymes share the CE-7signature motif. Accordingly, the perhydrolase catalyst of the presentinvention is an enzyme structurally classified as belonging to the CE-7carbohydrate esterase family. Each of the present perhydrolase enzymescomprise the CE-7 signature (diagnostic) motif.

Vincent et al. (supra) analyzed the structure CE-7 esterases and hasidentified several highly conserved motifs that are diagnostic for thefamily. As shown in FIG. 1, the highly conserved motifs (underlined inFIG. 1; position numbering relative to SEQ ID NO: 2) include theArg118-Gly119-Gln120 (RGQ), Gly179-Xaa180-Ser181-Gln182-Gly183 (GXSQG),and His298-Glu299 (HE). In addition, FIG. 1 illustrates an additionalhighly conserved Lys267-Xaa268-Asp269 (LXD) motif that may be used tofurther characterize the signature motif. All of the numbering isrelative to the numbering of a reference sequence (B. subtilisATCC31954™ perhydrolase; SEQ ID NO: 2).

In one embodiment, suitable perhydrolytic enzymes can be identified bythe presence of the CE-7 signature motif (Vincent et al., supra). In apreferred embodiment, perhydrolases comprising the CE-7 signature motifare identified using a CLUSTALW alignment against the Bacillus subtilisATCC 31954™ perhydrolase (SEQ ID NO: 2; i.e. the reference sequence usedfor relative amino acid position numbering). As per the amino acidresidue numbering of SEQ ID NO: 2, the CE-7 signature motif comprises

3 conserved motifs defined as:

-   -   a) Arg118-Gly119-Gln120;    -   a) Gly179-Xaa180-Ser181-Gln182-Gly183; and    -   b) His298-Glu299.        Typically, the Xaa at amino acid residue position 180 is glycine        or alanine. Amino acid residues belonging to the catalytic triad        are in bold.

Further analysis of the conserved motifs within the CE-7 carbohydrateesterase family indicates the presence of an additional conserved motif(LXD at amino acid positions 267-269 of SEQ ID NO: 2) that may be tofurther define a perhydrolase belonging to the CE-7 carbohydrateesterase family (FIGS. 1 a-c). In a further embodiment, the signaturemotif defined above includes a forth conserved motif defined as:

Leu267-Xaa268-Asp269.

The Xaa at amino acid residue position 268 is typically isoleucine,valine, or methionine. The forth motif includes the aspartic acidresidue (bold) belonging to the catalytic triad (Ser181-Asp269-His298).

Any number of well-known global alignment algorithms (i.e. sequenceanalysis software) may be used to align two or more amino acid sequences(representing enzymes having perhydrolase activity) to determine theexistence of the present signature motif (for example, CLUSTALW orNeedleman and Wunsch (J. Mol. Biol., 48:443-453 (1970)). The alignedsequence(s) is compared to the reference sequence (SEQ ID NO: 2). In oneembodiment, a CLUSTAL alignment (CLUSTALW) using a reference amino acidsequence (as used herein the CAH sequence (SEQ ID NO: 2) from theBacillus subtilis ATCC 31954™) is used to identify perhydrolasesbelonging to the CE-7 esterase family. The relative numbering of theconserved amino acid residues is based on the residue numbering of thereference amino acid sequence to account for small insertions ordeletions (5 amino acids or less) within the aligned sequence.

A comparison of the overall percent identity among perhydrolasesexemplified herein indicates that enzymes having as little as 42%identity to SEQ ID NO: 2 (while retaining the signature motif) exhibitsignificant perhydrolase activity and are structurally classified asCE-7 carbohydrate esterases. In one embodiment, the presentperhydrolases include enzymes comprising the present signature motif andat least 40%, preferably at least 42%, more preferably at least 50%,even more preferably at least 60%, yet even more preferably at least70%, even more preferably at least 80%, yet even more preferably atleast 90%, and most preferably at least 95% amino acid identity to SEQID NO: 2.

All of the present perhydrolases are comprised of the above signaturemotif as shown in Table 3.

TABLE 3 Conserved motifs found within the present enzymes havingperhydrolase activity. GXSQG HE motif^(a) Perhydrolase RGQ motif^(a)motif^(a) LXD motif^(b) (Residue Sequence (Residue #s) (Residue #s)Residue #s) #s) SEQ ID NO: 2 118-120 179-183 267-269 298-299 SEQ ID NO:6 118-120 179-183 267-269 298-299 SEQ ID NO: 8 118-120 179-183 267-269298-299 SEQ ID NO: 10 119-121 180-184 268-270 299-300 SEQ ID NO: 12118-120 179-183 267-269 298-299 SEQ ID NO: 14 119-121 181-185 269-271300-301 SEQ ID NO: 16 118-120 186-190 272-274 303-305 SEQ ID NO: 32118-120 179-183 267-269 298-299 ^(a)= Conserved motifs defined byVincent et al., supra used to define the signature motif. ^(b)= anadditional motif identified herein useful in further defining thesignature motif defined by Vincent et al., supra.

Alternatively, a contiguous signature motif (SEQ ID NO: 61) comprisingthe 4 conserved motifs (RGQ, GXSQG, LXD, and HE; Amino acids residues118-299 of SEQ ID NO: 2) may also be used as a contiguous signaturemotif to identify CE-7 carbohydrate esterases (FIGS. 1 a-c). As such,suitable enzymes expected to have perhydrolase activity may also beidentified as having at least 40%, preferably at least 50%, morepreferably at least 60%, more preferably at least 70%, more preferablyat least 80%, even more preferably at least 90%, and most preferably atleast 95% amino acid identity to SEQ ID NO: 61 (the 4 conserved motifsfound in CE-7 carbohydrate esterases are underlined).

(SEQ 1D NO: 61) RGQQSSEDTSISLHGHALGWMTKGILDKDTYYYRGVYLDAVRALEVISSFDEVDETRIGVTGGSQGGGLTIAAAALSDIPKAAVADYPYLSNFERAIDVALEQPYLEINSFFRRNGSPETEVQAMKTLSYFDIMNLADRVKVPVLMSIGLIDKVTPPSTVFAAYNHLETEKELKVYRYFGHE.

A comparison using the contiguous signature sequence against the presentCE-7 esterases having perhydrolase activity is provided in Table 4.BLASTP using default parameters was used.

TABLE 4 Percent Amino Acid Identity of Various CE-7 CarbohydrateEsterases having Perhydrolysis Activity Versus the Contiguous SignatureSequence (SEQ ID NO: 61). Perhydrolase % Identity using E-score SequenceBLASTP (expected) SEQ ID NO: 2 100 3e−92 SEQ ID NO: 6 98 6e−91 SEQ IDNO: 8 98 4e−98 SEQ ID NO: 10 78 1e−78 SEQ IDNO: 12 80 3e−76 SEQ ID NO:14 63 2e−56 SEQ ID NO: 16 51 1e−41 SEQ ID NO: 32 99 2e−90

Alternatively, the percent amino acid identity to the complete length ofone or more of the present perhydrolases may also be used. Accordingly,suitable enzymes having perhydrolase activity have at least 40%,preferably at least 42%, more preferably at least 50%, more preferablyat least 60%, more preferably at least 70%, even more preferably atleast 80%, yet even more preferably at least 90%, and most preferably atleast 95% amino acid identity to SEQ ID NO: 2. In a further embodiment,suitable perhydrolase catalysts comprise an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 8,SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, and SEQ IDNO: 32.

Suitable perhydrolase enzymes may also include enzymes having one ormore deletions, substitutions, and/or insertions to one of the presentperhydrolase enzymes (e.g. SEQ ID NOs 2, 6, 8, 10, 12, 14, 16, and 32).As shown in Table 3, CE-7 carbohydrates esterases having perhydrolaseactivity share as little as 42% overall amino acid identity. Based onthe data provided in the present examples, additional enzymes havingperhydrolase activity belonging to the CE-7 carbohydrate esterase familymay have even lower percent identity, so long as the enzyme retains theconserved signature motif. As such, the numbers of deletions,substitutions, and/or insertions may vary so long as the conservedsignature motifs (see Table 2) are found in their relative positionswithin enzyme.

Additionally, it is well within one of skill in the art to identitysuitable enzymes according to the structural similarity found within thecorresponding nucleic acid sequence. Hybridization techniques can beused to identity similar gene sequences. Accordingly, suitableperhydrolase catalysts of the present invention comprise an amino acidsequence encoded by a nucleic acid molecule that hybridizes understringent conditions to a nucleic acid molecule having a nucleic acidsequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO:15, SEQ ID NO: 31, SEQ ID NO: 41, and SEQ ID NO: 60.

The present method produces industrially-useful, efficaciousconcentrations of peracids in situ under aqueous reaction conditionsusing the perhydroiase activity of an enzyme belonging to the CE-7family of carbohydrate esterases. In one embodiment, the enzyme havingperhydrolase activity is also classified structurally and functionallyas a cephalosporin C deacetylase (CAH). In another embodiment, theenzyme having perhydrolase activity is classified structurally andfunctionally as an acetyl xylan esterase (AXE).

The peracids produced are quite reactive and generally decrease inconcentration over time. As such, it may be desirable to keep thevarious reaction components separated, especially for liquidformulations. In one aspect, the hydrogen peroxide source is separatefrom either the substrate or the perhydrolase catalyst, preferably fromboth. This can be accomplished using a variety of techniques including,but not limited to the use of multicompartment chambered dispensers(U.S. Pat. No. 4,585,150) and at the time of use physically combiningthe perhydrolase catalyst with an inorganic peroxide and the presentsubstrates to initiate the aqueous enzymatic perhydrolysis reaction. Theperhydrolase catalyst may optionally be immobilized within the body ofreaction chamber or separated (e.g. filtered, etc.) from the reactionproduct comprising the peracid prior to contacting the surface and/orobject targeted for treatment. The perhydrolase catalyst may be in aliquid matrix or in a solid form (i.e. powdered, tablet) or embeddedwithin a solid matrix that is subsequently mixed with the substrates toinitiate the enzymatic perhydrolysis reaction. In a further aspect, theperhydrolase catalyst may be contained within a dissolvable or porouspouch that may be added to the aqueous substrate matrix to initiateenzymatic perhydrolysis. In an additional further aspect, a powdercomprising the enzyme catalyst is suspended in the substrate (e.g.,triacetin), and at time of use is mixed with a source of peroxygen inwater.

HPLC Assay Method for Determining the Concentration of Peracid andHydrogen Peroxide.

A variety of analytical methods can be used in the present method toanalyze the reactants and products including, but not limited totitration, high performance liquid chromatography (HPLC), gaschromatography (GC), mass spectroscopy (MS), capillary electrophoresis(CE), the analytical procedure described by U. Karst et al., (Anal.Chem., 69(17):3623-3627 (1997)), and the2,2′-azino-bis(3-ethylbenzothazoline)-6-sulfonate (ABTS) assay (S.Minning, et al., Analytica Chimica Acta 378:293-298 (1999) and WO2004/058961 A1) as described in the present examples.

Determination of Minimum Biocidal Concentration of Peracids

The method described by J. Gabrielson, et al. (J. Microbiol. Methods 50:63-73 (2002)) can be employed for determination of the Minimum BiocidalConcentration (MBC) of peracids, or of hydrogen peroxide and enzymesubstrates. The assay method is based on XTT reduction inhibition, whereXTT((2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-5-[(phenylamino)carbonyl]-2H-tetrazolium,inner salt, monosodium salt) is a redox dye that indicates microbialrespiratory activity by a change in optical density (OD) measured at 490nm or 450 nm. However, there are a variety of other methods availablefor testing the activity of disinfectants and antiseptics including, butnot limited to viable plate counts, direct microscopic counts, dryweight, turbidity measurements, absorbance, and bioluminescence (see,for example Brock, Semour S., Disinfection, Sterilization, andPreservation, 5^(th) edition, Lippincott Williams & Wilkins,Philadelphia, Pa., USA; 2001).

Uses of Enzymatically-Prepared Peracid Compositions

The enzyme catalyst-generated peracid produced according to the presentmethods can be used in a variety of hard surface/inanimate objectapplications for reduction of concentrations of microbial, fungal,prion-related, and viral contamination, such as decontamination ofmedical instruments (e.g., endoscopes), textiles (e.g., garments,carpets), food preparation surfaces, food storage and food-packagingequipment, materials used for the packaging of food products, chickenhatcheries and grow-out facilities, animal enclosures, and spent processwaters that have microbial and/or virucidal activity. Theenzyme-generated peracids may be used in formulations designed toinactivate prions (e.g. certain proteases) to additionally providebiocidal activity. In a preferred aspect, the present peracidcompositions are particularly useful as a disinfecting agent fornon-autoclavable medical instruments and food packaging equipment. Asthe peracid-containing formulation may be prepared using GRAS orfood-grade components (enzyme, enzyme substrate, hydrogen peroxide, andbuffer), the enzyme-generated peracid may also be used fordecontamination of animal carcasses, meat, fruits and vegetables, or fordecontamination of prepared foods. The enzyme-generated peracid may beincorporated into a product whose final form is a powder, liquid, gel,film, solid or aerosol. The enzyme-generated peracid may be diluted to aconcentration that still provides an efficacious decontamination.

The compositions comprising an efficacious concentration of peracid canbe used to disinfect surfaces and/or objects contaminated (or suspectedof being contaminated) with viable pathogenic microbial contaminants bycontacting the surface or object with the products produced by thepresent processes. As used herein, “contacting” refers to placing adisinfecting composition comprising an effective concentration ofperacid in contact with the surface or inanimate object suspected ofcontamination with a disease-causing entity for a period of timesufficient to clean and disinfect. Contacting includes spraying,treating, immersing, flushing, pouring on or in, mixing, combining,painting, coating, applying, affixing to and otherwise communicating aperacid solution or composition comprising an efficacious concentrationof peracid, or a solution or composition that forms an efficaciousconcentration of peracid, with the surface or inanimate object suspectedof being contaminated with a concentration of a microbial population.The disinfectant compositions may be combined with a cleaningcomposition to provide both cleaning and disinfection. Alternatively, acleaning agent (e.g., a surfactant or detergent) may be incorporatedinto the formulation to provide both cleaning and disinfection in asingle composition.

The compositions comprising an efficacious concentration of peracid canalso contain at least one additional antimicrobial agent, combinationsof prion-degrading proteases, a virucide, a sporicide, or a biocide.Combinations of these agents with the peracid produced by the claimedprocesses can provide for increased and/or synergistic effects when usedto clean and disinfect surfaces and/or objects contaminated (orsuspected of being contaminated) with pathogenic microorganisms, spores,viruses, fungi, and/or prions. Suitable antimicrobial agents includecarboxylic esters (e.g., p-hydroxy alkyl benzoates and alkylcinnamates), sulfonic acids (e.g., dodecylbenzene sulfonic acid),iodo-compounds or active halogen compounds (e.g., elemental halogens,halogen oxides (e.g., NaOCl, HOCl, HOBr, ClO₂), iodine, interhalides(e.g., iodine monochloride, iodine dichloride, iodine trichloride,iodine tetrachloride, bromine chloride, iodine monobromide, or iodinedibromide), polyhalides, hypochlorite salts, hypochlorous acid,hypobromite salts, hypobromous acid, chloro- and bromo-hydantoins,chlorine dioxide, and sodium chlorite), organic peroxides includingbenzoyl peroxide, alkyl benzoyl peroxides, ozone, singlet oxygengenerators, and mixtures thereof, phenolic derivatives (e.g., o-phenylphenol, o-benzyl-p-chlorophenol, tert-amyl phenol and C₁-C₆ alkylhydroxy benzoates), quaternary ammonium compounds (e.g.,alkyldimethylbenzyl ammonium chloride, dialkyldimethyl ammonium chlorideand mixtures thereof), and mixtures of such antimicrobial agents, in anamount sufficient to provide the desired degree of microbial protection.Effective amounts of antimicrobial agents include about 0.001 wt % toabout 60 wt % antimicrobial agent, about 0.01 wt % to about 15 wt %antimicrobial agent, or about 0.08 wt % to about 2.5 wt % antimicrobialagent.

In one aspect, the peracids formed by the present process can be used toreduce the concentration of viable microbial contaminants (e.g. a viablemicrobial population) when applied on and/or at a locus. As used herein,a “locus” of the invention comprises part or all of a target surfacesuitable for disinfecting or bleaching. Target surfaces include allsurfaces that can potentially be contaminated with microorganisms,viruses, fungi, prions or combinations thereof. Non-limiting examplesinclude equipment surfaces found in the food or beverage industry (suchas tanks, conveyors, floors, drains, coolers, freezers, equipmentsurfaces, walls, valves, belts, pipes, drains, joints, crevasses,combinations thereof, and the like); building surfaces (such as walls,floors and windows); non-food-industry related pipes and drains,including water treatment facilities, pools and spas, and fermentationtanks; hospital or veterinary surfaces (such as walls, floors, beds,equipment, (such as endoscopes) clothing worn in hospital/veterinary orother healthcare settings, including clothing, scrubs, shoes, and otherhospital or veterinary surfaces); restaurant surfaces; bathroomsurfaces; toilets; clothes and shoes; surfaces of barns or stables forlivestock, such as poultry, cattle, dairy cows, goats, horses and pigs;hatcheries for poultry or for shrimp; and pharmaceutical orbiopharmaceutical surfaces (e.g., pharmaceutical or biopharmaceuticalmanufacturing equipment, pharmaceutical or biopharmaceuticalingredients, pharmaceutical or biopharmaceutical excipients). Additionalhard surfaces also include food products, such as beef, poultry, pork,vegetables, fruits, seafood, combinations thereof, and the like. Thelocus can also include water absorbent materials such as infected linensor other textiles. The locus also includes harvested plants or plantproducts including seeds, corms, tubers, fruit, and vegetables, growingplants, and especially crop growing plants, including cereals, leafvegetables and salad crops, root vegetables, legumes, berried fruits,citrus fruits and hard fruits.

Non-limiting examples of hard surface materials are metals (e.g., steel,stainless steel, chrome, titanium, iron, copper, brass, aluminum, andalloys thereof), minerals (e.g., concrete), polymers and plastics (e.g.,polyolefins, such as polyethylene, polypropylene, polystyrene,poly(meth)acrylate, polyacrylonitrile, polybutadiene,poly(acrylonitrile, butadiene, styrene), poly(acrylonitrile, butadiene),acrylonitrile butadiene; polyesters such as polyethylene terephthalate;and polyamides such as nylon). Additional surfaces include brick, tile,ceramic, porcelain, wood, vinyl, linoleum, and carpet.

Recombinant Microbial Expression

The genes and gene products of the instant sequences may be produced inheterologous host cells, particularly in the cells of microbial hosts.Preferred heterologous host cells for expression of the instant genesand nucleic acid molecules are microbial hosts that can be found withinthe fungal or bacterial families and which grow over a wide range oftemperature, pH values, and solvent tolerances. For example, it iscontemplated that any of bacteria, yeast, and filamentous fungi maysuitably host the expression of the present nucleic acid molecules. Theperhydrolase may be expressed intracellularly, extracellularly, or acombination of both intracellularly and extracellularly, whereextracellular expression renders recovery of the desired protein from afermentation product more facile than methods for recovery of proteinproduced by intracellular expression. Transcription, translation and theprotein biosynthetic apparatus remain invariant relative to the cellularfeedstock used to generate cellular biomass; functional genes will beexpressed regardless. Examples of host strains include, but are notlimited to bacterial, fungal or yeast species such as Aspergillus,Trichoderma, Saccharomyces, Pichia, Phaffia, Candida, Hansenula,Yarrowia, Salmonella, Bacillus, Acinetobacter, Zymomonas, Agrobacterium,Erythrobacter, Chlorobium, Chromatium, Flavobacterium, Cytophaga,Rhodobacter, Rhodococcus, Streptomyces, Brevibacterium, Corynebacteria,Mycobacterium, Deinococcus, Escherichia, Erwinia, Pantoea, Pseudomonas,Sphingomonas, Methylomonas, Methylobacter, Methylococcus, Methylosinus,Methylomicrobium, Methylocystis, Alcaligenes, Synechocystis,Synechococcus, Anabaena, Thiobacillus, Methanobacterium, Klebsiella, andMyxococcus. In one embodiment, bacterial host strains includeEscherichia, Bacillus, and Pseudomonas. In a preferred embodiment, thebacterial host cell is Escherichia coli.

Large-scale microbial growth and functional gene expression may use awide range of simple or complex carbohydrates, organic acids andalcohols or saturated hydrocarbons, such as methane or carbon dioxide inthe case of photosynthetic or chemoautotrophic hosts, the form andamount of nitrogen, phosphorous, sulfur, oxygen, carbon or any tracemicronutrient including small inorganic ions. The regulation of growthrate may be affected by the addition, or not, of specific regulatorymolecules to the culture and which are not typically considered nutrientor energy sources.

Vectors or cassettes useful for the transformation of suitable hostcells are well known in the art. Typically the vector or cassettecontains sequences directing transcription and translation of therelevant gene, a selectable marker, and sequences allowing autonomousreplication or chromosomal integration. Suitable vectors comprise aregion 5′ of the gene which harbors transcriptional initiation controlsand a region 3′ of the DNA fragment which controls transcriptionaltermination. It is most preferred when both control regions are derivedfrom genes homologous to the transformed host cell and/or native to theproduction host, although such control regions need not be so derived.

Initiation control regions or promoters, which are useful to driveexpression of the present cephalosporin C deacetylase coding region inthe desired host cell are numerous and familiar to those skilled in theart. Virtually any promoter capable of driving these genes is suitablefor the present invention including but not limited to CYC1, HIS3, GAL1,GAL10, ADH1, PGK, PHO5, GAPDH, ADC1, TRP1, URA3, LEU2, ENO, TPI (usefulfor expression in Saccharomyces); AOX1 (useful for expression inPichia); and lac, ara, tet, trp, IP_(L), IP_(R), T7, tac, and trc(useful for expression in Escherichia coli) as well as the amy, apr, nprpromoters and various phage promoters useful for expression in Bacillus.

Termination control regions may also be derived from various genesnative to the preferred host cell. In one embodiment, the inclusion of atermination control region is optional. In another embodiment, thechimeric gene includes a termination control region derived thepreferred host cell.

Industrial Production

A variety of culture methodologies may be applied to produce the presentperhydrolase catalysts. For example, large-scale production of aspecific gene product overexpressed from a recombinant microbial hostmay be produced by both batch and continuous culture methodologies.

A classical batch culturing method is a closed system where thecomposition of the media is set at the beginning of the culture and notsubject to artificial alterations during the culturing process. Thus, atthe beginning of the culturing process, the media is inoculated with thedesired organism or organisms and growth or metabolic activity may occurwithout adding anything further to the system. Typically, however, a“batch” culture is batch with respect to the addition of carbon sourceand attempts are often made to control factors such as pH and oxygenconcentration. In batch systems the metabolite and biomass compositionsof the system change constantly up to the time the culture isterminated. Within batch cultures cells moderate through a static lagphase to a high growth log phase and finally to a stationary phase wheregrowth rate is diminished or halted. If untreated, cells in thestationary phase will eventually die. Cells in log phase are oftenresponsible for the bulk of production of end product or intermediate insome systems. Stationary or post-exponential phase production can beobtained in other systems.

A variation on the standard batch system is the fed-batch system.Fed-batch culture processes are also suitable in the present inventionand comprise a typical batch system except that the substrate is addedin increments as the culture progresses. Fed-batch systems are usefulwhen catabolite repression is apt to inhibit the metabolism of the cellsand where it is desirable to have limited amounts of substrate in themedia. Measurement of the actual substrate concentration in fed-batchsystems is difficult and is estimated on the basis of the changes ofmeasurable factors such as pH, dissolved oxygen and the partial pressureof waste gases such as CO₂. Batch and fed-batch culturing methods arecommon and well known in the art and examples may be found in Thomas D.Brock in Biotechnology: A Textbook of Industrial Microbiology, SecondEdition, Sinauer Associates, Inc., Sunderland, Mass. (1989) andDeshpande, Mukund V., Appl. Biochem. Biotechnol., 36:227 (1992).

Commercial production of the desired products may also be accomplishedwith a continuous culture. Continuous cultures are an open system wherea defined culture media is added continuously to a bioreactor and anequal amount of conditioned media is removed simultaneously forprocessing. Continuous cultures generally maintain the cells at aconstant high liquid phase density where cells are primarily in logphase growth. Alternatively, continuous culture may be practiced withimmobilized cells where carbon and nutrients are continuously added, andvaluable products, by-products or waste products are continuouslyremoved from the cell mass. Cell immobilization may be performed using awide range of solid supports composed of natural and/or syntheticmaterials.

Continuous or semi-continuous culture allows for the modulation of onefactor or any number of factors that affect cell growth or end productconcentration. For example, one method will maintain a limiting nutrientsuch as the carbon source or nitrogen level at a fixed rate and allowall other parameters to moderate. In other systems a number of factorsaffecting growth can be altered continuously while the cellconcentration, measured by media turbidity, is kept constant. Continuoussystems strive to maintain steady state growth conditions and thus thecell loss due to media being drawn off must be balanced against the cellgrowth rate in the culture. Methods of modulating nutrients and growthfactors for continuous culture processes as well as techniques formaximizing the rate of product formation are well known in the art ofindustrial microbiology and a variety of methods are detailed by Brock,supra.

Fermentation media in the present invention must contain suitable carbonsubstrates. Suitable substrates may include but are not limited tomonosaccharides such as glucose and fructose, disaccharides such aslactose or sucrose, polysaccharides such as starch or cellulose ormixtures thereof and unpurified mixtures from renewable feedstocks suchas cheese whey permeate, cornsteep liquor, sugar beet molasses, andbarley malt. Additionally, the carbon substrate may also be one-carbonsubstrates such as carbon dioxide, methane or methanol (for example,when the host cell is a methylotrophic microorganism). Similarly,various species of Candida will metabolize alanine or oleic acid (Sulterat al., Arch. Microbiol., 153:485-489 (1990)). Hence, it is contemplatedthat the source of carbon utilized in the present invention mayencompass a wide variety of carbon-containing substrates and will onlybe limited by the choice of organism.

Applicants specifically incorporate the entire contents of all citedreferences in this disclosure. Further, when an amount, concentration,or other value or parameter is given either as a range, preferred range,or a list of upper preferable values and lower preferable values, thisis to be understood as specifically disclosing all ranges formed fromany pair of any upper range limit or preferred value and any lower rangelimit or preferred value, regardless of whether ranges are separatelydisclosed. Where a range of numerical values is recited herein, unlessotherwise stated, the range is intended to include the endpointsthereof, and all integers and fractions within the range. It is notintended that the scope of the invention be limited to the specificvalues recited when defining a range.

GENERAL METHODS

The following examples are provided to demonstrate preferred aspects ofthe invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

All reagents and materials were obtained from DIFCO Laboratories(Detroit, Mich.), GIBCO/BRL (Gaithersburg, Md.), TCI America (Portland,Oreg.), Roche Diagnostics Corporation (Indianapolis, Ind.) orSigma/Aldrich Chemical Company (St. Louis, Mo.), unless otherwisespecified.

The following abbreviations in the specification correspond to units ofmeasure, techniques, properties, or compounds as follows: “sec” or “s”means second(s), “min” means minute(s), “h” or “hr” means hour(s), “μL”means microliters, “mL” means milliliters, “L” means liters, “mM” meansmillimolar, “M” means molar, “mmol” means millimole(s), “ppm” meansparts per million, “wt” means weight, “wt %” means weight percent, “g”means grams, “μg” means micrograms, “g” means gravity, “HPLC” means highperformance liquid chromatography, “dd H₂O” means distilled anddeionized water, “dcw” means dry cell weight, “ATCC” or “ATCC®” meansthe American Type Culture Collection (Manassas, Va.), “U” means units ofperhydrolase activity, “rpm” means revolutions per minute, and “EDTA”means ethylenediaminetetraacetic acid.

EXAMPLE 1 Growth of Bacillus subtilis ATCC 31954™ and Preparation ofCell Extract

A culture of Bacillus subtilis (ATCC 31954™) was revived followingsuspension of the dried culture in 5 mL of nutrient broth (DIFCO;0003-01-6) and incubation for 3 days at 30° C. Following the third dayof incubation, an aliquot of the culture was streaked onto a trypticasesoy agar culture plate (Becton, Dickinson, and Company; Franklin Lakes,N.J.) and incubated at 35° C. for 24 h. Several single colonies werescraped onto a 1 microliter inoculation loop (Becton Dickinson; catalog#220215) and transferred into 50 mL of Lactobacillus MRS broth (HardyDiagnostics, Santa Maria, Calif.; catalog #C5931). The culture was thengrown at 30° C. and a 200-rpm agitation rate for 12 h. After 12 h ofgrowth, 2 mL of the culture was transferred into an unbaffled 500-mLshake flask containing 100 mL of MRS broth for growth at 30° C. and200-rpm agitation for 12-14 h. The cells were subsequently harvested bycentrifugation at 15,000×g for 25 min at 5° C. and the resulting cellpaste stored at −80° C.

For cell extract preparation, 0.9 g of cell paste was suspended at 25 wt% (wet cell weight) in 0.05 M potassium phosphate buffer (pH 7.0)containing dithiothreitol (1 mM) and EDTA (1 mM). The cell suspensionwas passed twice through a French press having a working pressure of16,000 psi. The crude extract was then centrifuged at 20,000×g to removecellular debris, producing a clear cell extract that was assayed fortotal soluble protein (Bicinchoninic Acid Kit for Protein Determination,Sigma Aldrich, Sigma catalog #BCA1-KT), then frozen and stored at −80°C.

EXAMPLE 2 Determination of Perhydrolysis Activity of Bacillus subtilisATCC 31954™ Semi-Purified Cell Extract

A 1.0-mL aliquot of Bacillus subtilis (ATCC 31954™) cell extract (10 mgtotal protein/mL, prepared as described in Example 1) was diluted withan equal volume of 50 mM phosphate buffer (pH 7.0) and filtered througha 100,000 Molecular Weight Cutoff (MWCO) Centricon membrane unit(Millipore Corp, Bedford, Mass.). The resulting filtrate (semi-purifiedcell extract) contained 1.5 mg total protein/mL assayed for totalsoluble protein (Bicinchoninic Acid Kit for Protein Determination, Sigmacatalog #BCA-KT), and an assay of this filtrate indicated no measurablecatalase activity.

A 1-mL reaction mixture containing triacetin (250 mM), hydrogen peroxide(2.5 M) and 0.100 mL of semi-purified cell extract (0.15 mg extracttotal protein) in 50 mM phosphate buffer (pH 6.5) was mixed at 25° C. Acontrol reaction was run by substituting 50 mM phosphate buffer (pH 6.5)for semi-purified cell extract to determine the concentration ofperacetic acid produced by chemical perhydrolysis of triacetin byhydrogen peroxide in the absence of added semi-purified cell extract.

Determination of the concentration of peracetic acid in the reactionmixture was performed according to the method described by Karst et al.Aliquots (0.250 mL) of the reaction mixture were removed at 10 min and30 min and filtered using an Ultrafree® MC-filter unit (30,000 NormalMolecular Weight Limit (NMWL), Millipore cat #UFC3LKT 00) bycentrifugation for 2 min at 12,000 rpm; removal of the protein componentof the aliquot by filtration terminated the reaction. An aliquot (0.100mL) of the resulting filtrate was transferred to 1.5-mL screw cap HPLCvial (Agilent Technologies, Palo Alto, Calif.; #5182-0715) containing0.300 mL of deionized water, then 0.100 mL of 20 mM MTS(methyl-p-tolyl-sulfide) in acetonitrile was added, the vials capped,and the contents briefly mixed prior to a 10 min incubation at ca. 25°C. in the absence of light. To each vial was then added 0.400 mL ofacetonitrile and 0.100 mL of a solution of triphenylphosphine (TPP, 40mM) in acetonitrile, the vials re-capped, and the resulting solutionmixed and incubated at ca. 25° C. for 30 min in the absence of light. Toeach vial was then added 0.100 mL of 10 mM N,N-diethyl-m-toluamide(DEET; HPLC external standard) and the resulting solution analyzed byHPLC as described below. The peracetic acid concentrations produced in10 min and 30 min is listed in Table 5.

HPLC Method:

Supelco Discovery C8 column (10-cm×4.0-mm, 5 μm) (cat. #569422-U)w/precolumn Supelco Supelguard Discovery C8 (Sigma-Aldrich; cat#59590-U); 10 microliter injection volume; gradient method with CH₃CN(Sigma-Aldrich; #270717) and deionized H₂O at 1.0 mL/min and ambienttemperature:

Time (min:sec) (% CH₃CN) 0:00 40 3:00 40 3:10 100 4:00 100 4:10 40 7:00(stop) 40

TABLE 5 Peracetic acid (PAA) produced by reaction of triacetin (250 mM)and hydrogen peroxide (2.5M) at pH 6.5 in the presence or absence of B.subtilis (ATCC 31954 ™) semi-purified cell extract. B. subtilis (ATCC31954 ™) semi-purified cell extract peracetic acid (ppm) peracetic acid(ppm) (mg total protein/mL) in 10 min in 30 min 0 641 1343 0.15 34923032

EXAMPLE 3 Perhydrolysis Activity of Semi-Purified Enzyme from Bacillussubtilis ATCC 31954™ Cell Extract

Bacillus subtilis ATCC 31954™ growth and extract preparation wasperformed as described in Example 1, except that the crude extract wasnot centrifuged. The crude extract was fractionated with cold n-propanol(−20° C.). A flask containing the cell-free extract was stirred in anice bath for 15 min, then the n-propanol (−20° C.) was added drop-wise(to prevent freezing of the extract) to a concentration of 40% (v/v).The resulting extract/propanol mixture was stirred in the ice bath for30 min, then centrifuged at 12,000×g for 10 min at 5° C., and thesupernatant returned to the flask and placed into the ice bath.Additional n-propanol (−20° C.) was slowly added to the supernatant withstirring to a concentration of 60% (v/v), and the resulting mixturestirred for 30 min in the ice bath and then centrifuged as before. Thepellet from this second fraction was saved on ice and the supernatantreturned to the flask and placed into the ice bath. Cold n-propanol wasslowly added to the supernatant with stirring to a concentration of 80%(v/v), the mixture stirred for 30 min and centrifuged as before. Thepellet from the 60-80% fraction was saved on ice. The pellets from the40-60% (v/v) n-propanol fractions and the 60-80% (v/v) n-propanolfractions were dissolved in a minimum amount of 0.05 M phosphate buffer(pH 6.5) and the resulting solutions assayed for total soluble protein(Bicinchoninic Acid Kit for Protein Determination, catalog #BCA1-KT),then frozen and stored at −80° C.

A 1-mL reaction mixture containing triacetin (250 mM), hydrogen peroxide(1.0 M) and 0.10 mg/mL of total soluble protein from either the 40-60%(v/v) or 60-80% (v/v) n-propanol fractions of the cell extract (preparedas described above) in 50 mM phosphate buffer (pH 6.5) was mixed at 25°C. A control reaction was run by substituting 50 mM phosphate buffer (pH6.5) for the n-propanol fractions of the cell extract containingsemi-purified enzyme to determine the concentration of peracetic acidproduced by chemical perhydrolysis of triacetin by hydrogen peroxide inthe absence of added semi-purified enzyme. The reaction mixture wasassayed for peracetic acid at 5 min and 30 min using the proceduredescribed in Example 2, and the concentrations of peracetic acidproduced by added enzyme are listed in Table 6.

TABLE 6 Peracetic acid (PAA) produced by reaction of triacetin (250 mM)and hydrogen peroxide (1.0M) at pH 6.5 in the presence or absence of B.subtilis (ATCC 31954 ™) semi-purified cell extracts. peracetic acidn-propanol fraction total protein peracetic acid (ppm) of cell extract(mg/mL reaction) (ppm) in 5 min in 30 min no extract 0 221 803 40-60%0.1 2829 4727 60-80% 0.1 1832 3777

EXAMPLE 4 Identification of a Cephalosporin C Deacetylase HavingPerhydrolysis Activity from Bacillus subtilis ATCC 31954™ Cell Extract

A 0.1 mL sample (500 μg total protein) of the 40-60% n-propanol fractiondescribed in Example 3 was mixed at room temperature with an equalvolume of 2× non-denaturing (native) sample buffer (Invitrogen) andloaded into the preparative sample well of a 1.5 mm 8-16% Tris-Glycinepolyacrylamide mini-gel (2D gels; Invitrogen). The native gelelectrophoresis was operated at 125 V for 90 min using Tris-Glycinerunning buffer (Invitrogen). Following electrophoresis, the gel wasprepared for an in situ esterase activity assay using the pH indicator,bromothymol blue.

The gel was washed for 10 min×2 with deionized water and slow mechanicalmixing. The gel was then washed for 10 min using 10 mM phosphate buffer.Following the removal of the phosphate buffer, 50 mL of 10 mM phosphatebuffer containing 665 μL of saturated bromothymol blue (in water) wasincubated with the gel for 10 min followed by the addition of 1 mL ofneat triacetin (Sigma Aldrich). Within 10 min of incubation one yellowband at 146 kDa appeared on the gel indicating esterase activity.

The esterase-positive band was excised from the gel and transferred intoa 50 mL polypropylene conical tube (Falcon). The yellow bromothymol bluestain was removed from the gel slice following two 5-mL deionized waterwashes with gentle mixing. The gel slice was then treated for 30 minwith 0.9 mL of 2× Novex Tris-Glycine SDS sample buffer plus 100 μL of10× NuPAGE reducing agent (Invitrogen) with gentle mixing. Following thesample treatment, the gel slice and sample buffer were incubated at 85°C. for 5 min using a hot water bath. The gel slice was then removed fromthe incubation tube and carefully placed in the single preparative wellof a 1.5 mm 8-16% Tris-Gly mini-gel. Care was taken to exclude airbubbles and to have direct contact with the stacking gel. The gel slicewas then immobilized in place following the addition of 250-300 μL of awarm 0.5% agarose solution prepared in deionized water into thepreparative well. The single molecular marker lane was loaded with 15 μLof SeeBlue® Plus2 pre-stained MW marker (Invitrogen).

The electrophoresis of the gel slice was operated at 30 V for 30 min forelectro-elution of the protein from the gel slice into the slab gel. Thevoltage was then ramped up from 30 V to 125 V over 10 min followed by 90min operation at 125 V. Following electrophoresis, the resolved proteinbands on the gel were blotted onto a PVDF membrane as described in theXCell II™ blotting manual (Invitrogen) and the blotting buffer was 10 mMCAPS, pH 11.0. The electro-blotting procedure was operated at 25 V for 2hr at room temperature with ice water in the jacket of the transferapparatus.

Following the transfer, the PVDF membrane was stained with ProBlotstaining solution (Applied Biosystems, Foster City, Calif.) for 1 minfollowed by de-staining with methanol:water (50:50). Six protein bandswere identified and each was N-terminal sequenced. Following a Blastsearch of the GenBank® amino acid sequence database, the only bandhaving esterase-related sequence homology was identified as Band 1 andthe 17 N-terminal amino acid calls had 100% amino acid identity to aBacillus subtilis cephalosporin C deacetylase (GENBANK® BAA01729;Mitsushima et al., supra; U.S. Pat. No. 5,528,152; and U.S. Pat. No.5,338,676).

EXAMPLE 5 Cloning and Expression of Perhydrolase from Bacillus subtilisATCC 31954™

Genomic DNA was isolated from Bacillus subtilis ATCC 31954™ using thePUREGENE® DNA purification system (Gentra Systems, Minneapolis Minn.).The perhydrolase gene was amplified from the genomic DNA by PCR (0.5 minat 94° C., 0.5 min at 55° C., 1 min at 70° C., 30 cycles) using primersidentified as SEQ ID NO: 3 and SEQ ID NO: 4. The resulting nucleic acidproduct (SEQ ID NO: 1) was subcloned into pTrcHis2-TOPO® (Invitrogen,Carlsbad Calif.) to generate the plasmid identified as pSW186. Theperhydrolase gene was also amplified from the genomic DNA by PCR (0.5min at 94° C., 0.5 min at 55° C., 1 min at 70° C., 30 cycles) usingprimers identified as SEQ ID NO: 27 and SEQ ID NO: 28. The resultingnucleic acid product (SEQ ID NO: 29) was cut with restriction enzymesPstI and XbaI and subcloned between the PstI and XbaI sites in pUC19 togenerate the plasmid identified as pSW194. The plasmids pSW186 andpSW194 were used to transform E. coli TOP10 (Invitrogen, CarlsbadCalif.), E. coli MG1655 (ATCC 47076™), E. coli UM2 (E. coli GeneticStock Center #7156, Yale University, New Haven Conn.) and E. coli KLP18(see EXAMPLE 15) to generate the strains identified as TOP10/pSW186,MG1655/pSW186, UM2/pSW186, KLP18/pSW186, TOP10/pSW194, MG1655/pSW194,UM2/pSW194 and KLP18/pSW194, respectively. TOP10/pSW186, MG1655/pSW186,UM2/pSW186, KLP18/pSW186, TOP10/pSW194, MG1655/pSW194, UM2/pSW194 andKLP18/pSW194 were gown in LB media at 37° C. with shaking up toOD_(600 nm)=0.4-0.5, at which time IPTG was added to a finalconcentration of 1 mM, and incubation continued for 2-3 h. Cells wereharvested by centrifugation and SOS-PAGE was performed to confirmexpression of the perhydrolase at 20-40% of total soluble protein.

EXAMPLE 6 Cloning and Expression of Perhydrolase from Bacillus subtilisBE1010

Genomic DNA was isolated from Bacillus subtilis BE1010 (Payne andJackson 1991 J. Bacteria 173:2278-2282) using the PUREGENE® DNApurification system (Gentra Systems). The perhydrolase gene wasamplified from the genomic DNA by PCR (0.5 min at 94° C., 0.5 min at 55°C., 1 min at 70° C., 30 cycles) using primers identified as SEQ ID NO: 3and SEQ ID NO: 4. The resulting nucleic acid product (SEQ ID NO: 5) wassubcloned into pTrcHis2-TOPO® (Invitrogen) to generate the plasmididentified as pSW187. The perhydrolase gene was also amplified from thegenomic DNA by PCR (0.5 min at 94° C., 0.5 min at 55° C., 1 min at 70°C., 30 cycles) using primers identified as SEQ ID NO: 27 and SEQ ID NO:28. The resulting nucleic acid product (SEQ ID NO: 30) was cut withrestriction enzymes PstI and XbaI and subcloned between the PstI andXbaI sites in pUC19 to generate the plasmid identified as pSW189. Theplasmids pSW187 and pSW189 were used to transform E. coli TOP10(Invitrogen), E. coli MG1655 (ATCC 47076™), E. coli UM2 (E. coli GeneticStock Center #7156, Yale University, New Haven Conn.) and E. coli KLP18(see EXAMPLE 15) to generate the strains identified as TOP101pSW187,MG1655/pSW187, UM2/pSW187, KLP181pSW187, TOP101pSW189, MG1655/pSW189,UM2/pSW189 and KLP18/pSW19, respectively. TOP101pSW187, MG1655/pSW187,UM2/pSW187, KLP18/pSW187, TOP10/pSW189, MG1655/pSW189, UM2/pSW189 andKLP18/pSW189 were gown in LB media at 37° C. with shaking up toOD_(600 nm)=0.4-0.5, at which time IPTG was added to a finalconcentration of 1 mM, and incubation continued for 2-3 h. Cells wereharvested by centrifugation and SDS-PAGE was performed to confirmexpression of the perhydrolase at 20-40% of total soluble protein.

EXAMPLE 7 Cloning and Expression of Perhydrolase from Bacillus subtilisATCC 6633™

Genomic DNA was isolated from Bacillus subtilis ATCC 6633™ using thePUREGENE® DNA purification system. The perhydrolase gene was amplifiedfrom the genomic DNA by PCR (0.5 min at 94° C., 0.5 min at 55° C., 1 minat 70° C., 30 cycles) using primers identified as SEQ ID NO: 3 and SEQID NO: 4. The resulting nucleic acid product (SEQ ID NO: 7) wassubcloned into pTrcHis2-TOPO® to generate the plasmid identified aspSW188. The plasmid pSW188 was used to transform E. coli MG1655 (ATCC47076™) and E. coli UM2 (E. coli Genetic Stock Center #7156, YaleUniversity, New Haven Conn.) to generate the strains identified asMG1655/pSW188 and UM2/pSW188, respectively. MG1655/pSW188 and UM2/pSW188were gown in LB media at 37° C. with shaking up to OD_(600 nm)=0.4-0.5,at which time IPTG was added to a final concentration of 1 mM, andincubation continued for 2-3 h. Cells were harvested by centrifugationand SDS-PAGE was performed to confirm expression of the perhydrolase at20-40% of total soluble protein.

EXAMPLE 8 Cloning and Expression of Perhydrolase from Bacillus subtilisATCC 29233™

Genomic DNA was isolated from Bacillus subtilis ATCC 29233™ using thePUREGENE® DNA purification system. The perhydrolase gene was amplifiedfrom the genomic DNA by PCR (0.5 min at 94° C., 0.5 min at 55° C., 1 minat 70° C., 30 cycles) using primers identified as SEQ ID NO: 3 and SEQID NO: 4. The resulting nucleic acid product (SEQ ID NO: 31) wassubcloned into pTrcHis2-TOPO® to generate the plasmid identified aspSW190. The plasmid pSW190 was used to transform E. coli MG1655 (ATCC47076™), E. coli UM2 (E. coli Genetic Stock Center #7156, YaleUniversity, New Haven Conn.) and E. coli KLP18 (see EXAMPLE 15) togenerate the strains identified as MG1655/pSW190, UM2/pSW190 andKLP18/pSW190, respectively. MG16551pSW190, UM2/pSW190 and KLP18/pSW190were gown in LB media at 37° C. with shaking up to OD_(600 nm)=0.4-0.5,at which time IPTG was added to a final concentration of 1 mM, andincubation continued for 2-3 h. Cells were harvested by centrifugationand SDS-PAGE was performed to confirm expression of the perhydrolase at20-40% of total soluble protein.

EXAMPLE 9 Cloning and Expression of Perhydrolase from Bacilluslicheniformis ATCC 14580™

Genomic DNA was isolated from Bacillus licheniformis ATCC 14580™ usingthe PUREGENE® DNA purification system. The perhydrolase gene wasamplified from the genomic DNA by PCR (0.5 min at 94° C., 0.5 min at 55°C., 1 min at 70° C., 30 cycles) using primers identified as SEQ ID NO:33 and SEQ ID NO: 34. The resulting nucleic acid product (SEQ ID NO: 9)was subcloned into pTrcHis2-TOPO® to generate the plasmid identified aspSW191. The plasmid pSW191 was used to transform E. coli MG1655 (ATCC47076™), E. coli UM2 (E. coli Genetic Stock Center #7156, YaleUniversity, New Haven Conn.), E. coli PIR1 (Invitrogen, Carlsbad Calif.)and E. coli KLP18 (see EXAMPLE 15) to generate the strains identified asMG1655/pSW191, UM2/pSW191, PIR1/pSW191 and KLP18/pSW191, respectively.MG1655/pSW191, UM21pSW191, PIR1/pSW191 and KLP18/pSW191 were gown in LBmedia at 37° C. with shaking up to OD_(500 nm)=0.4-0.5, at which timeIPTG was added to a final concentration of 1 mM, and incubationcontinued for 2-3 h. Cells were harvested by centrifugation and SDS-PAGEwas performed to confirm expression of the perhydrolase at 20-40% oftotal soluble protein.

EXAMPLE 10 Cloning and Expression of Perhydrolase from Clostridiathermocellum ATCC 27405™

Genomic DNA was isolated from Clostridia thermocellum ATCC 27405™ usingthe PUREGENE® DNA purification system. The perhydrolase gene wasamplified from the genomic DNA by PCR (0.5 min at 94° C., 0.5 min at 55°C., 1 min at 70° C., 30 cycles) using primers identified as SEQ ID NO:35 and SEQ ID NO: 36. The resulting nucleic acid product (SEQ ID NO: 13)was subcloned into pTrcHis2-TOPO® to generate the plasmid identified aspSW193. The plasmid pSW193 was used to transform E. coli MG1655 (ATCC47076™), E. coli UM2 (E. coli Genetic Stock Center #7156, YaleUniversity, New Haven Conn.) and E. coli KLP18 (see EXAMPLE 15) togenerate the strains identified as MG1655/pSW193, UM2/pSW193 andKLP18/pSW193, respectively. MG1655/pSW193, UM2/pSW193 and KLP18/pSW193were gown in LB media at 37° C. with shaking up to OD_(600 nm)=0.4-0.5,at which time IPTG was added to a final concentration of 1 mM, andincubation continued for 2-3 h. Cells were harvested by centrifugationand SOS-PAGE was performed to confirm expression of the perhydrolase at20-40% of total soluble protein.

EXAMPLE 11 Cloning and Expression of Perhydrolase from Bacillus pumilusPS213

The gene encoding acetyl xylan esterase (axe1) from B. pumilus PS213 asreported in GENBANK® (accession #AJ249957) was synthesized using codonsoptimized for expression in E. coli (DNA 2.0, Menlo Park Calif.). Thegene was subsequently amplified by PCR (0.5 min at 94° C., 0.5 min at55° C., 1 min at 70° C. 30 cycles) using primers identified as SEQ IDNO: 37 and SEQ ID NO: 38. The resulting nucleic acid product (SEQ ID NO:60) was subcloned into pTrcHis2-TOPO® (Invitrogen, Carlsbad Calif.) togenerate the plasmid identified as pSW195. The plasmid pSW195 was usedto transform E. coli MG1655 (ATCC 47076™), E. coli UM2 (E. coli GeneticStock Center #7156, Yale University, New Haven Conn.) and E. coli KLP18(see EXAMPLE 15) to generate the strains identified as MG1655/pSW195,UM2/pSW195 and KLP18/pSW195, respectively. MG1655/pSW195, UM2/pSW195 andKLP18/pSW195 were gown in LB media at 37° C. with shaking up to OD600nm=0.4-0.5, at which time IPTG was added to a final concentration of 1mM, and incubation continued for 2-3 h. Cells were harvested bycentrifugation and SDS-PAGE was performed to confirm expression of theperhydrolase at 20-40% of total soluble protein.

EXAMPLE 12 Cloning and Expression of Perhydrolase from Thermotoganeapolitana

The gene encoding acetyl xylan esterase from Thermotoga neapolitana asreported in GENBANK® (accession #58632) was synthesized using codonsoptimized for expression in E. coli (DNA 2.0, Menlo Park, Calif.). Thegene was subsequently amplified by PCR (0.5 min at 94° C., 0.5 min at55° C., 1 min at 70° C., 30 cycles) using primers identified as SEQ IDNO: 39 and SEQ ID NO: 40. The resulting nucleic acid product (SEQ ID NO:41) was subcloned into pTrcHis2-TOPO® to generate the plasmid identifiedas pSW196. The plasmid pSW196 was used to transform E. coli MG1655 (ATCC47076™), E. coli UM2 (E. coli Genetic Stock Center #7156, YaleUniversity, New Haven Conn.) and E. coli KLP18 (see EXAMPLE 15) togenerate the strains identified as MG1655/pSW196, UM2/pSW196 andKLP18/pSW196, respectively. MG16551pSW196, UM2/pSW196 And KLP18/pSW196were gown in LB media at 37° C. with shaking up to OD600 nm=0.4-0.5, atwhich time IPTG was added to a final concentration of 1 mM, andincubation continued for 2-3 h. Cells were harvested by centrifugationand SDS-PAGE was performed to confirm expression of the perhydrolase at20-40% of total soluble protein.

EXAMPLE 13 Construction of a katG Catalase Disrupted E. coli Strain

The kanamycin resistance gene (kan; SEQ ID NO: 42) was amplified fromthe plasmid pKD13 (SEQ ID NO: 43) by PCR (0.5 min at 94° C., 0.5 min at55° C., 1 min at 70° C., 30 cycles) using primers identified as SEQ IDNO: 44 and SEQ ID NO: 45 to generate the PCR product identified as SEQID NO: 46. The katG nucleic acid sequence is provided as SEQ ID NO: 47and the corresponding amino acid sequence is SEQ ID NO: 48. E. coliMG1655 (ATCC 47076™) was transformed with the temperature-sensitiveplasmid pKD46 (SEQ ID NO: 49), which contains the A-Red recombinasegenes (Datsenko and Wanner, 2000, PNAS USA 97:6640-6645), and selectedon LB-amp plates for 24 h at 30° C. MG1655/pKD46 was transformed with50-500 ng of the PCR product by electroporation (BioRad Gene Pulser, 0.2cm cuvette, 2.5 kV, 200 W, 25 uF), and selected on LB-kan plates for 24h at 37° C. Several colonies were streaked onto LB-kan plates andincubated overnight at 42° C. to cure the pKD46 plasmid. Colonies werechecked to confirm a phenotype of kanR/ampS. Genomic DNA was isolatedfrom several colonies using the PUREGENE® DNA purification system, andchecked by PCR to confirm disruption of the katG gene using primersidentified as SEQ ID NO: 50 and SEQ ID NO: 51. Several katG-disruptedstrains were transformed with the temperature-sensitive plasmid pCP20(SEQ ID NO: 52), which contains the FLP recombinase, used to excise thekan gene, and selected on LB-amp plates for 24 h at 37° C. Severalcolonies were streaked onto LB plates and incubated overnight at 42° C.to cure the pCP20 plasmid. Two colonies were checked to confirm aphenotype of kanS/ampS, and called MG1655 KatG1 and MG1655 KatG2.

EXAMPLE 14

Construction of a katE Catalase Disrupted E. coli Strain

The kanamycin resistance gene (SEQ ID NO: 42) was amplified from theplasmid pKD13 (SEQ ID NO: 43) by PCR (0.5 min at 94° C., 0.5 min at 55°C., 1 min at 70° C., 30 cycles) using primers identified as SEQ ID NO:53 and SEQ ID NO: 54 to generate the PCR product identified as SEQ IDNO: 55. The katE nucleic acid sequence is provided as SEQ ID NO: 56 andthe corresponding amino acid sequence is SEQ ID NO: 57. E. coli MG1655(ATCC 47076 ™) was transformed with the temperature-sensitive plasmidpKD46 (SEQ ID NO: 49), which contains the A-Red recombinase genes, andselected on LB-amp plates for 24 h at 30° C. MG1655/pKD46 wastransformed with 50-500 ng of the PCR product by electroporation (BioRadGene Pulser, 0.2 cm cuvette, 2.5 kV, 200 W, 25 uF), and selected onLB-kan plates for 24 h at 37° C. Several colonies were streaked ontoLB-kan plates and incubated overnight at 42° C. to cure the pKD46plasmid. Colonies were checked to confirm a phenotype of kanR/ampS.Genomic DNA was isolated from several colonies using the PUREGENE DNApurification system, and checked by PCR to confirm disruption of thekatE gene using primers identified as SEQ ID NO: 58 and SEQ ID NO: 59.Several katE-disrupted strains were transformed with thetemperature-sensitive plasmid pCP20 (SEQ ID NO: 52), which contains theFLP recombinase, used to excise the kan gene, and selected on LB-ampplates for 24 h at 37° C. Several colonies were streaked onto LB platesand incubated overnight at 42° C. to cure the pCP20 plasmid. Twocolonies were checked to confirm a phenotype of kanS/ampS, and calledMG1655 KatE1 and MG1655 KatE2

EXAMPLE 15 Construction of a katG Catalase and katE Catalase DisruptedE. coli Strain (KLP18)

The Kanamycin Resistance Gene (SEQ ID NO: 42) was Amplified from theplasmid pKD13 (SEQ ID NO: 43) by PCR (0.5 min at 94 CC, 0.5 min at 55°C., 1 min at 70° C., 30 cycles) using primers identified as SEQ ID NO:53 and SEQ ID NO: 54 to generate the PCR product identified as SEQ IDNO: 55. E. coli MG1655 KatG1 (EXAMPLE 13) was transformed with thetemperature-sensitive plasmid pKD46 (SEQ ID NO: 49), which contains theλ-Red recombinase genes, and selected on LB-amp plates for 24 h at 30°C. MG1655 KatG1/pKD46 was transformed with 50-500 ng of the PCR productby electroporation (BioRad Gene Pulser, 0.2 cm cuvette, 2.5 kV, 200 W,25 uF), and selected on LB-kan plates for 24 h at 37° C. Severalcolonies were streaked onto LB-kan plates and incubated overnight at 42°C. to cure the pKD46 plasmid. Colonies were checked to confirm aphenotype of kanR/ampS. Genomic DNA was isolated from several coloniesusing the PUREGENE® DNA purification system, and checked by PCR toconfirm disruption of the katE gene using primers identified as SEQ IDNO: 58 and SEQ ID NO: 59. Several katE-disrupted strains (Δ katE) weretransformed with the temperature-sensitive plasmid pCP20 (SEQ ID NO:52), which contains the FLP recombinase, used to excise the kan gene,and selected on LB-amp plates for 24 h at 37° C. Several colonies werestreaked onto LB plates and incubated overnight at 42° C. to cure thepCP20 plasmid. Two colonies were checked to confirm a phenotype ofkanS/ampS, and called MG1655 KatG1KatE18.1 and MG1655 KatG1KatE23.MG1655 KatG1KatE18.1 is designated E. coli KLP18.

EXAMPLE 16 Estimation of Perhydrolase Molecular Mass

Cell pellets obtained from shake flask growths of E. coli KLP18, acatalase double knockout of E. coli MG1655, expressing perhydrolasegenes from Bacillus subtilis, Bacillus licheniformis and Clostridiumthermocellum, were suspended in 2.2 mL of 0.05 M potassium phosphatebuffer (pH 7.0) containing dithiothreitol (1 mM). Each cell suspensionwas passed twice through a French press having a working pressure of16,000 psi (˜110.3 MPa). The crude extracts were centrifuged at 20,000×gto remove cellular debris, producing clear crude extracts that wereassayed for total soluble protein (Bicinchoninic Acid Kit [BCA] forProtein Determination, Sigma Aldrich, BCA1-KT).

Clarified crude extracts (5 μL) containing 20 μg total protein weremixed at room temperature with an equal volume of 2× non-denaturing(native) sample buffer (Invitrogen) and loaded into sample wells of a1.5 mm×10 well 4-12% Tris-Glycine polyacrylamide mini-gel (Invitrogen),and 7.5 μL of NATIVEMARK™ Unstained Protein Standard (Invitrogen) wasloaded into two separate wells. Native gel electrophoresis was performedat 125 V for 105 min using Tris-Glycine running buffer (Invitrogen).Following electrophoresis, the gel was prepared for an in situ esteraseactivity assay using the pH indicator bromothymol blue.

The gel was washed for 10 min×2 with deionized water and slow mechanicalmixing. The gel was then washed for 10 min using 10 mM pH 7.0 phosphatebuffer and slow mechanical mixing. Following the removal of thephosphate buffer, 30 mL of 10 mM pH 7.0 phosphate buffer containing 400μL of saturated bromothymol blue in water was incubated with the gel for10 min followed by the addition of 1 mL of neat triacetin (TessenderloFine Chemicals; Staffordshire, UK). Within 2 minutes of incubationyellow bands developed at the active perhydrolase enzyme sites. All B.subtilis species and B. licheniformis had intense bands around amolecular mass of 216 kDa. The C. thermocellum displayed an intensemajor primary band around 432 kDa and a minor secondary band around 576kDa, indicating esterase activity. All bands were marked by punching asmall hole in the gel adjacent to the bands. The gel was washed for 10min×2 with deionized water and slow mechanical mixing to remove theesterase activity stain. The gel was then washed for 10 min using 10 mMphosphate buffer with slow mechanical mixing to prepare for proteinstaining. Coomassie blue stain was added to cover the gel. After 5minutes of slow mechanical mixing, the Coomassie blue was decanted andreplaced with 40 mL de-stain (10% acetic acid, 30% methanol, 60%de-ionized water). After de-staining, the molecular masses of the activeareas were estimated. The results are summarized in Table 7.

TABLE 7 Estimation of Perhydrolase Molecular Mass. Primary Secondarynative gel native gel activity activity stain, stain, Calculatedestimated estimated sub-unit molecular molecular molecular TransformantPerhydrolase mass mass mass strain source (kDa) (kDa) (kDa) KLP18 nonenone none — KLP18/pSW186 B. subtilis 216 none 35.8 ATCC 31954 ™ detectedKLP18/pSW189 B. subtilis 216 none 35.9 BE1010 detected KLP18/pSW190 B.subtilis 216 none 35.8 ATCC 29233 ™ detected KLP18/pSW191 B.licheniformis 216 none 35.8 ATCC14580 ™ detected KLP18/pSW193 C.thermocellum 432 648 36.0 ATCC 27405 ™

EXAMPLE 17 Fermentation of E. coli UM2/pSW187 Expressing B. subtilisBE1010 Perhydrolase

A fermenter seed culture was prepared by charging a 2-L shake flask with0.5 L seed medium containing LB Miller medium (DIFCO). The pH of themedium was adjusted to 6.8 and sterilized in the flask.Post-sterilization, 1 mL of ampicillin stock solution (25 mg/mL) wasadded. The seed medium was inoculated with a 1-mL culture of E. coliUM2/pSW187 in 20% glycerol, and cultivated at 36° C. and 300 rpm. Theseed culture was transferred at ca. 1-2 OD₅₅₀ to a 14 L fermentor(Braun) with 8 L of medium at 35° C. containing KH₂PO₄ (3.50 g/L), FeSO₄heptahydrate (0.05 g/L), MgSO₄ heptahydrate (2.0 g/L), sodium citratedihydrate (1.90 g/L), yeast extract (Ambrex 695, 5.0 g/L), Biospumex153Kantifoam (0.25 mL/L, Cognis Corporation), NaCl (1.0 g/L), CaCl₂dihydrate (10 g/L), and NIT trace elements solution (10 mL/L). The traceelements solution contained citric acid monohydrate (10 g/L), MnSO₄hydrate (2 g/L), NaCl (2 g/L), FeSO₄ heptahydrate (0.5 g/L), ZnSO₄heptahydrate (0.2 g/L), CuSO₄ pentahydrate (0.02 g/L) and NaMoO₄dihydrate (0.02 g/L). Post sterilization addition included 60 g fedbatch solution (see below) and 16.0 mL ampicillin stock solution (25mg/mL). A fed-batch solution contained 2.4 kg of 60% w/w glucose, 0.6 Lof 25 g/L yeast extract and 50 g/L Bacto peptone (DIFCO). Glucose feedwas initiated when the glucose concentration decreased below 0.5 g/L,starting at 0.3 g/min, and increased progressively each hour to 0.35,0.40, 0.47, and 0.53 g/min, respectively; the rate remained constantafterwards. Glucose concentration in the medium was monitored and if theconcentration exceeded 0.1 g/L the addition rate was decreased orstopped temporarily. Induction was initiated at OD₅₅₀=7 with theaddition of 16 mL IPTG (0.5 M). The temperature was controlled at 36°C., the aeration was fixed at 2 slpm (standard liters per minute) withagitation at 400 rpm. The pH was controlled at 6.8; NH₄OH (29% w/w) andH₂SO₄ (20% w/v) were used for pH control. The head pressure was 0.5 bar.The cells were harvested by centrifugation at 8 h post IPTG addition.

EXAMPLE 18 Fermentation of E. coli UM2/pSW186 Expressing B. subtilisATCC 31954™ Perhydrolase or E. coli UM2/pSW191 Expressing B.licheniformis ATCC 14580™ Perhydrolase

The seed culture was prepared as described in Example 17 using E. coliUM2/pSW186 expressing B. subtilis ATCC31954™ perhydrolase or E. coliUM2/pSW191 expressing B. licheniformis ATCC14580™ perhydrolase. Thefermentation medium was LB Miller (25 g/L, DIFCO). Post sterilizationadditions included 50 g glucose solution (50% w/w) and 16.0 mLampicillin stock solution (25 mg/mL). Glucose (50% w/w) was used for fedbatch fermentation. Glucose feed was initiated when the glucoseconcentration decreased below 0.5 g/L, at a constant rate of 0.3 g/min.Glucose concentration in the medium was monitored and if theconcentration exceeded 0.1 g/L the addition rate was decreased orstopped temporarily. Induction was initiated at OD₅₅₀=2 with addition of16 mL IPTG (0.5 M). The temperature was controlled at 36° C., theaeration was fixed at 2 slpm with agitation at 400 rpm. The pH wascontrolled at 6.8; NH₄OH (29% w/w) and H₂SO₄ (20% w/v) were used for pHcontrol. The head pressure was 0.5 bar. The cells were harvested bycentrifugation at 8 h post IPTG addition.

EXAMPLE 19 Fermentation of E. coli KLP181PSW189 Expressing B. subtilisBE1010 Perhydrolase or E. coli KLP18/PSW191 Expressing B. licheniformisATCC 14580™ Perhydrolase

A fermentor seed culture was prepared by charging a 2-L shake flask with0.5 L seed medium containing yeast extract (Amberx 695, 5.0 g/L), K₂HPO₄(10.0 g/L), KH₂PO₄ (7.0 g/L), sodium citrate dihydrate (1.0 g/L),(NH₄)₂SO₄ (4.0 g/L), MgSO₄ heptahydrate (1.0 g/L) and ferric ammoniumcitrate (0.10 g/L). The pH of the medium was adjusted to 6.8 and themedium was sterilized in the flask. Post sterilization additionsincluded glucose (50 wt %, 10.0 mL) and 1 mL ampicillin (25 mg/mL) stocksolution. The seed medium was inoculated with a 1-mL culture of E. coliKLP18/PSW189 or KLP181PSW191 in 20% glycerol, and cultivated at 35° C.and 300 rpm. The seed culture was transferred at ca. 1-2 OD₅₅₀ to a 14 Lfermentor (Braun) with 8 L of medium at 35° C. containing KH₂PO₄ (3.50g/L), FeSO₄ heptahydrate (0.05 g/L), MgSO₄ heptahydrate (2.0 g/L),sodium citrate dihydrate (1.90 g/L), yeast extract (Ambrex 695, 5.0g/L), Biospumex153K antifoam (0.25 mL/L, Cognis Corporation), NaCl (1.0g/L), CaCl₂ dihydrate (10 g/L), and NIT trace elements solution (10g/L). The trace elements solution contained citric acid monohydrate (10g/L), MnSO₄ hydrate (2 g/L), NaCl (2 g/L), FeSO₄ heptahydrate (0.5 g/L),ZnSO₄ heptahydrate (0.2 g/L), CuSO₄ pentahydrate (0.02 g/L) and NaMoO₄dihydrate (0.02 g/L). Post sterilization additions included glucosesolution (50% w/w, 80.0 g) and ampicillin (25 mg/mL) stock solution(16.00 mL). Glucose solution (50% w/w) was used for fed batch. Glucosefeed was initiated when glucose concentration decreased to 0.5 g/L,starting at 0.31 g feed/min and increasing progressively each hour to0.36, 0.42, 0.49, 0.57, 0.66, 0.77, 0.90, 1.04, 1.21, 1.41 1.63 g/minrespectively; the rate remained constant afterwards. Glucoseconcentration in the medium was monitored and if the concentrationexceeded 0.1 g/L the feed rate was decreased or stopped temporarily. ForE. coli KLP18/PSW191, the induction was initiated at OD₅₅₀=80 withaddition of 16 mL IPTG (0.5 M), for E. coli KLP18/PSW189 the growth wasslower and induction was initiated at OD₅₅₀=56. The dissolved oxygen(DO) concentration was controlled at 25% of air saturation. The DO wascontrolled first by impeller agitation rate (400 to 1400 rpm) and laterby aeration rate (2 to 10 slpm). The pH was controlled at 6.8. NH₄OH(29% w/w) and H₂SO₄ (20% w/v) were used for pH control. The headpressure was 0.5 bars. The cells were harvested by centrifugation 16 hpost IPTG addition.

EXAMPLE 20 E. coli KLP18 Versus E. coli UM2 as Fermentation Host forPerhydrolase Production

E. coli KLP18 (EXAMPLE 15) was used to produce transformants (EXAMPLES5, 8, 9 and 10) that were grown in multiple 10-L fermentations followingthe method described in EXAMPLE 19. The final OD for these runs iscompared to fermentations that produced E. coli UM2 transformants(EXAMPLES 5, 8, 9 and 10) expressing these same perhydrolases that wererun following the fermentation methods described in EXAMPLES 17 and 18.Table 8 summarizes 10-L fermentation runs with both UM2 and KLP18 ashost, and demonstrates the superior performance of KLP18 compared toUM2.

TABLE 8 run time, final run ID host plasmid perhydrolase (h) OD₅₅₀ PAA25UM2 pSW186 SEQ ID NO: 2 21.6 11.9 PAA26 UM2 pSW186 SEQ ID NO: 2 7.4 11.9PAA42 UM2 pSW186 SEQ ID NO: 2 12.4 5.5 PAA43 UM2 pSW186 SEQ ID NO: 212.4 5.5 PAA48 KLP18 pSW186 SEQ ID NO: 2 33.1 181.0 PAA30 UM2 pSW190 SEQID NO: 32 12.1 6.2 PAA31 UM2 pSW190 SEQ ID NO: 32 12.3 8.8 PAA40 UM2pSW190 SEQ ID NO: 32 12.7 4.6 PAA41 UM2 pSW190 SEQ ID NO: 32 12.6 5.3PAA49 KLP18 pSW190 SEQ ID NO: 32 33.6 128.0 PAA39 UM2 pSW191 SEQ ID NO:10 10.6 6.5 PAA46 KLP18 pSW191 SEQ ID NO: 10 33.6 140.0 PAA50 KLP18pSW191 SEQ ID NO: 10 36.2 155.0 PAA45 UM2 pSW193 SEQ ID NO: 14 12.4 5.7PAA51 KLP18 pSW193 SEQ ID NO: 14 35.7 147.0

EXAMPLE 21 Evaluation of Bacillus subtilis ATCC 31954™ PerhydrolaseExpressed in E. coli Transformants

The three transformants E. coli TOP10/pSW186, E. coli MG1655/pSW186 andE. coli UM2/pSW186 described in Example 5 were grown in unbaffled shakeflasks containing Miller's LB broth (50 mL; Mediatech, Inc, Herndon,Va.) with ampicillin (100 μg/mL) for 14-16 h at 35-37° C. with 200 rpmagitation. Following the overnight growth of the three transformants,each culture was sub-cultured by preparing a 1:100 dilution of eachculture into fresh Miller's LB broth containing ampicillin (100 μg/mL).Following a 3 h growth at 35-37° C. with 200 rpm agitation, each culturewas induced by the addition of IPTG to a final concentration of 1 mM.After an additional 3 h growth under the same conditions, the cell pastefrom each culture was harvested by centrifugation at 26,000×g for 20 minat 5° C. Cell extracts of each of the transformants were preparedaccording to the procedure described in Example 1, except that theextraction buffer used to prepare the 25 wt % wet cell suspension wascomposed of 0.05 M potassium phosphate (pH 7.0) and 1 mM dithiothreitol.

Separate 1-mL reactions containing triacetin (250 mM), hydrogen peroxide(1.0 M) and 50 μg of extract total protein from one of the three cellextracts (prepared as described above) in 50 mM phosphate buffer (pH6.5) were run at 25° C. A control reaction was run by substituting 50 mMphosphate buffer (pH 6.5) for the extract total protein solution todetermine the concentration of peracetic acid produced by chemicalperhydrolysis of triacetin by hydrogen peroxide in the absence of addedextract protein. A second set of control reactions was run using 50 μgof extract total protein prepared from extracts of untransformed E. coliTOP10, E. coli MG1655 and E. coli UM2 to determine the background levelof peracid produced by each strain in the absence of expressedperhydrolase. The concentration of peracetic acid in the reactionmixtures was determined according to the method of Karst et al.described in Example 2 (Table 9).

TABLE 9 Peracetic acid (PAA) produced by reaction of triacetin (250 mM)and hydrogen peroxide (1.0M) at pH 6.5 in the presence of cell extractsof E. coli TOP10/pSW186, E. coli MG1655/pSW186 and E. coli UM2/pSW186.total protein total protein (□g/mL peracetic acid peracetic acid extractsource reaction) (ppm) in 5 min (ppm) in 30 min no extract 0 188 598TOP10 50 181 654 TOP10/pSW186 50 2684 5363 MG1655 50 173 638MG1655/pSW186 50 1354 4333 UM2 50 175 655 UM2/pSW186 50 3002 6529

EXAMPLE 22 Perhydrolytic Activity of E. coli TOP10/pSW186 ExtractExpressing Bacillus subtilis ATCC 31954™ Perhydrolase

Separate 1.0 mL triacetin perhydrolysis reactions were run as describedin Example 21 using the E. coli TOP10/pSW186 transformant extract toprovide one of the following total protein concentrations in thereaction: 196 μg/mL, 98 μg/mL, 49 μg/mL, 25 μg/mL, 12.5 μg/mL, 6.25μg/mL, 3.0 μg/mL, or 1.5 μg/mL total protein concentration in eachreaction (Table 10).

TABLE 10 Dependence of peracetic acid (PAA) concentration on totalprotein concentration derived from E. coli TOP10/pSW186 transformantextract in reactions containing triacetin (250 mM) and hydrogen peroxide(1.0M) at pH 6.5. total protein total protein (□g/mL peracetic acidperacetic acid extract source reaction) (ppm) in 5 min (ppm) in 30 minno extract 0 193 854 TOP10 50 181 654 TOP10/pSW186 1.5 580 1710TOP10/pSW186 3.0 824 2233 TOP10/pSW186 6.3 1371 3029 TOP10/pSW186 12.52052 4587 TOP10/pSW186 25 2849 4957 TOP10/pSW186 49 4294 TOP10/pSW186 984244 TOP10/pSW186 196 4294

EXAMPLE 23 Perhydrolytic Activity of E. coli UM2/pSW186 ExtractExpressing Bacillus subtilis ATCC 31954™ Perhydrolase

An extract of E. coli UM2/pSW186 transformant (20 mg total protein/mLextract, prepared as described in Example 21) was employed in 1.0 mLperhydrolysis reactions (run as described in Example 21) containingtriacetin (40 mM or 100 mM), hydrogen peroxide (40 mM or 100 mM) andextract total protein (0.1 mg/mL or 1.0 mg/mL) in phosphate buffer (Pi,100 mM, 200 mM or 300 mM) at pH 6.5 or 7.5 at 25° C. each reaction(Table 11).

TABLE 11 Dependence of peracetic acid (PAA) concentration on triacetinand hydrogen peroxide concentrations using perhydrolase derived from E.coli UM2/pSW186 transformant extract at pH 6.5 or 7.5. total proteinH₂O₂ triacetin Pi PAA (ppm) PAA (ppm) (mg/mL) (mM) (mM) (mM) pH in 5 minin 30 min 0 40 40 100 6.5 0 0 0 40 100 100 6.5 0 0 0.1 40 40 100 6.5 490 1 40 40 100 6.5 239 160 1 40 100 100 6.5 439 560 0 40 100 200 6.5 0 00 100 100 200 6.5 1 30 0 100 100 200 7.5 14 1 0 100 100 300 7.5 5 4 1100 40 200 6.5 75 9 1 100 100 200 6.5 1150 925 1 40 100 200 7.5 290 80 1100 100 300 7.5 332 58

EXAMPLE 24 Evaluation of Perhydrolase Expressed in E. coli TransformantsDerived from Bacillus subtilis BE1010

The E. coli TOP10/pSW187, E. coli MG1655/pSW187 and E. coli UM21pSW187transformants described in Example 6 were grown in unbaffled shakeflasks containing Miller's LB broth (50 mL; Mediatech, Inc, Herndon,Va.) with ampicillin (100 μg/mL) for 14-16 h at 35-37° C. with 200 rpmagitation. Following the overnight growth of the three transformants,each culture was sub-cultured by preparing a 1:100 dilution of eachculture into fresh Miller's LB broth containing ampicillin (100 μg/mL).Following a 3 hour growth at 35-37° C. with 200 rpm agitation, eachculture was induced by the addition of IPTG to a final concentration of1 mM. After an additional 3 hours growth under the same conditions, thecell paste from each culture was harvested by centrifugation at 26,000×gfor 20 min at 5° C. For cell extract preparation, the proceduredescribed in Example 1 was repeated except that the extraction bufferused to prepare the 25 wt % wet cell suspension was composed of 0.05 Mpotassium phosphate (pH 7.0) and 1 mM dithiothreitol.

Separate 1.0 mL reactions containing triacetin (250 mM), hydrogenperoxide (1.0 M) and 50 μg of extract total protein in 50 mM phosphatebuffer (pH 6.5) were run at 25° C. with each transformant extract. Acontrol reaction was run substituting 50 mM phosphate buffer (pH 6.5)for the extract total protein solution to determine the concentration ofperacetic acid produced by chemical perhydrolysis of triacetin withhydrogen peroxide. A second set of control reactions was run using 50 μgof extract total protein prepared from extracts of untransformed E. coliTOP10, E. coli MG1655 and E. coli UM2 to determine the background levelof peracid produced by each strain in the absence of expressedperhydrolase. The concentration of peracetic acid in the reactionmixtures (Table 12) was determined according to the method of Karst etal., as described in Example 2.

TABLE 12 Peracetic acid (PAA) produced by reaction of triacetin (250 mM)and hydrogen peroxide (1.0M) at pH 6.5 in the presence of cell extractsof E. coli TOP10/pSW187, E. coli MG1655/pSW187 and E. coli UM2/pSW187.total protein total protein peracetic acid peracetic acid extract source(□g/mL reaction) (ppm) in 5 min (ppm) in 30 min no extract 0 159 626TOP10 50 181 654 TOP10/pSW187 50 3192 6663 MG1655 50 173 638MG1655/pSW187 50 3472 7349 UM2 50 175 655 UM2/pSW187 50 3741 7626

EXAMPLE 25 Evaluation of Perhydrolases Expressed in E. coliTransformants

The transformants were prepared as described in Examples 5, 6, 7, 8, 9,10, 18 and 19. Cell extracts of each of the transformants were preparedaccording to the procedure described in Example 1, except that theextraction buffer used to prepare the 25 wt % wet cell suspension wascomposed of 0.05 M potassium phosphate (pH 7.0) and 1 mM dithiothreitol.

Separate 1-mL reactions containing triacetin (250 mM), hydrogen peroxide(1.0 M) and 50 μg of extract total protein from a cell extract (preparedas described above) in 50 mM phosphate buffer (pH 6.5) were run at 25°C. A control reaction was run by substituting 50 mM phosphate buffer (pH6.5) for the extract total protein solution to determine theconcentration of peracetic acid produced by chemical perhydrolysis oftriacetin by hydrogen peroxide in the absence of added extract protein.A second set of control reactions was run using 50 μg of extract totalprotein prepared from extracts of untransformed E. coli TOP10, E. coliMG1655, E. coli UM2 and E. coli KLP18 to determine the background levelof peracid produced by each strain in the absence of expressedperhydrolase. The concentration of peracetic acid in the reactionmixtures (Table 13) was determined according to the method of Karst etal. described in Example 2.

TABLE 13 Peracetic acid (PAA) produced by reaction of triacetin (250 mM)and hydrogen peroxide (1.0M) at pH 6.5 in the presence of 50 μg of totalextract protein/mL from transformant cell extracts of E. coli TOP10, E.coli MG1655, E. coli UM2, E. coli PIR2, and E. coli KLP18. PAA PAA (ppm)(ppm) transformant PAA 5 min, PAA 30 min, cell perhydrolase (ppm) no(ppm) no extract source 5 min extract 30 min extract TOP10 none(control) 181 188 654 598 MG1655 none (control) 173 188 638 598 UM2 none(control) 175 188 655 598 PIR2 none (control) 144 276 515 677 KLP18 none(control) 200 100 555 330 TOP10/ B. subtilis 2684 188 5363 598 pSW186ATCC 31954 ™ MG1655/ B. subtilis 1354 188 4333 598 pSW186 ATCC 31954 ™UM2/pSW186 B. subtilis 3002 188 6529 598 ATCC 31954 ™ KLP18/ B. subtilis1033 268 2641 792 pSW186 ATCC 31954 ™ TOP10/ B. subtilis 3192 159 6663626 pSW187 BE1010 MG1655/ B. subtilis 3472 159 7349 626 pSW187 BE1010UM2/pSW187 B. subtilis 3741 159 7626 626 BE1010 KLP18/ B. subtilis 2631146 6579 625 pSW189 BE1010 UM2/pSW188 B. subtilis 4617 289 8742 306 ATCC6633 ™ UM2/pSW190 B. subtilis 5314 320 8845 738 ATCC 29233 ™ UM2/pSW190aB. subtilis 2622 234 3553 642 ATCC 29233 ™ KLP18/ B. subtilis 1006 1463285 625 pSW190 ATCC 29233 ™ PIR2/pSW191 B. licheniformis 3125 276 6338677 ATCC 14580 ™ UM2/ B. licheniformis 1632 276 4640 677 pSW191 ATCC14580 ™ KLP18/ B. licheniformis 3936 146 8016 625 pSW191 ATCC 14580 ™MG1655/ C. thermocellum 2279 349 3178 645 pSW193 ATCC 27405 ™ UM2/pSW193C. thermocellum 2738 349 3597 645 ATCC 27405 ™ KLP18/ C. thermocellum1687 146 2407 625 pSW193 ATCC 27405 ™ UM2/pSW195 B. pumilus 2226 3606354 776 PS213 KLP18/ B. pumilus 5023 100 9642 394 pSW195 PS213UM2/pSW196 T. neapolitana 1347 360 2553 776 KLP18/ T. neapolitana 878100 2023 394 pSW196

EXAMPLE 26 Comparative Evaluation of Commercial Lipases forPerhydrolysis

Separate 1-mL reactions containing triacetin (250 mM), hydrogen peroxide(1.0 M) and 50 μg of commercial lipases in 50 mM phosphate buffer (pH6.5) were run at 25° C. Control reactions were run without commerciallipase to determine the concentration of peracetic acid produced bychemical perhydrolysis of triacetin by hydrogen peroxide in the absenceof added lipase. The concentration of peracetic acid in the reactionmixtures (Table 14) was determined according to the method of Karst etal. described in Example 2. The commercial lipases were obtained fromSigma/Aldrich Chemical Company (St. Louis, Mo.), BioCatalytics(Pasadena, Calif.), Meito Sangyo Co. (Nagoya, Japan), Amano Enzymes(Lombard, Ill.), Novozymes (Franklinton, N.C.), Valley Research (SouthBend, Ind.), and Enzyme Development Corporation (ENZECO®; New York,N.Y.).

TABLE 14 Peracetic acid (PAA) produced by reaction of triacetin (250 mM)and hydrogen peroxide (1.0M) at pH 6.5 in the presence of 50 μg/mL ofcommercial lipases. PAA PAA (ppm); (ppm); commercial lipase lipasesource 5 min 30 min no enzyme control 105 105 Meito MY Candida rugosa155 280 Meito OF Candida rugosa 120 340 Meito AL Achromobacter sp. 165315 Meito PL Alcaligines sp. 165 430 Meito SL Pseudomonas cepacia 210440 Meito TL Pseudomonas stutzeri 225 500 Meito QLC Alcaligines sp. 195240 Meito QLM Alcaligines sp. 225 555 no enzyme control 150 205 AmanoF-DS Rhizopus oryzae 180 265 Amano R Penicillium roqueforti 170 160Amano M 10 Mucor javanicus 255 425 Amano G 50 Penicillium cambertii 4040 Amano F-AP15 Rhizopus oryzae 120 50 Amano AY 30 Candida rugosa 140300 Amano PS Burkholder cepacia 150 150 Amano DS Aspergillus niger 140125 Amano AY Candida rugosa 180 390 Amano AK-20 Pseudomonas fluorescens215 500 Amano LPS Burkholder cepacia 315 350 Amano A 12 Aspergillusniger 245 490 no enzyme control 30 55 BioCatalytics ICR 110 Candidaantartica B 145 245 Novozymes Lipolase Thermomyces 10 0 100 L type EXlanuginosus Novozymes Lipozyme Thermomyces 125 370 TL 100 L lanuginosusNovozymes Lipozyme Candida antartica 0 180 CALB L Novozymes PalataseAspergillus oryzae 95 220 20000L Valley Research CR Candida rugosa 70320 Valley Research MJ Mucor javanicus 140 440 Valley Research ANAspergillus niger 165 240 Enzeco LC Candida rugosa 105 120 Enzeco MLCAspergillus niger 140 370 Enzeco R0 20 Rhizopus oryzae 55 100

EXAMPLE 27 Evaluation of Perhydrolases Expressed in E. coliTransformants

Cell extracts of transformants expressing perhydrolase were preparedaccording to the procedure described in Example 21. Separate 1-mLreactions containing triacetin (105 mM), hydrogen peroxide (78 mM) and 1mg or 2 mg of extract total protein from a cell extract (prepared asdescribed above) in 50 mM phosphate buffer (pH 6.5) were run at 25° C. Acontrol reaction was run by substituting 50 mM phosphate buffer (pH 6.5)for the extract total protein solution to determine the concentration ofperacetic acid produced by chemical perhydrolysis of triacetin byhydrogen peroxide in the absence of added extract protein. Theconcentration of peracetic acid in the reaction mixtures (Table 15) wasdetermined according to the method of Karst et al. described in Example2.

TABLE 15 Peracetic acid (PAA) produced by reaction of triacetin (105 mM)and hydrogen peroxide (78 mM) at pH 6.5 or 7.5 in the presence of 1 mgor 2 mg of total extract protein/mL from transformant cell extracts ofE. coli MG1655, E. coli UM2, E. coli PIR2 and E. coli KLP18. total PAAPAA transformant cell source of protein (ppm); (ppm); extractperhydrolase (mg/mL) pH 5 min 30 min no extract control 0 6.5 0 0 noextract control 0 7.5 8 12 UM2/pSW186 B. subtilis ATCC 1.0 6.5 945 142031954 ™ UM2/pSW186 B. subtilis ATCC 2.0 6.5 1000 1250 31954 ™ UM2/pSW186B. subtilis ATCC 1.0 7.5 1001 1215 31954 ™ UM2/pSW186 B. subtilis ATCC2.0 7.5 1036 1050 31954 ™ no extract control 0 6.5 0 0 no extractcontrol 0 7.5 45 0 MG1655/pSW187 B. subtilis 1.0 6.5 690 265 BE1010UM2/pSW187 B. subtilis 1.0 6.5 730 755 BE1010 UM2/pSW187 B. subtilis 2.06.5 1400 1990 BE1010 UM2/pSW187 B. subtilis 2.0 7.5 1710 2105 BE1010KLP18/pSW189 B. subtilis 1.0 6.5 885 1288 BE1010 KLP18/pSW189 B.subtilis 2.0 6.5 950 1263 BE1010 no extract control 0 6.5 0 0 UM2/pSW190B. subtilis ATCC 1.0 6.5 940 685 29233 ™ no extract control 0 6.5 0 0PIR2/pSW191 B. lichen. ATCC 1.0 6.5 860 1305 14580 ™ UM2/pSW191 B.lichen. ATCC 1.0 6.5 675 1530 14580 ™ no extract control 0 6.5 0 0UM2/pSW195 B. pumilus 1.0 6.5 400 850 PS213 UM2/pSW195 B. pumilus 2.06.5 460 790 PS213 no extract control 0 6.5 0 0 UM2/pSW196 T. neapolitana1.0 6.5 1100 1685 UM2/pSW196 T. neapolitana 2.0 6.5 1190 1900

EXAMPLE 28 Comparative Evaluation of Commercial Lipases forPerhydrolysis

Separate 1-mL reactions containing triacetin (105 mM), hydrogen peroxide(78 mM) and 1 mg of commercial lipases in 50 mM phosphate buffer (pH6.5) were run at 25° C. A control reaction was run without commerciallipase to determine the concentration of peracetic acid produced bychemical perhydrolysis of triacetin by hydrogen peroxide in the absenceof added lipase. The concentration of peracetic acid in the reactionmixtures (Table 16) was determined according to the method of Karst etal. described in Example 2.

TABLE 16 Peracetic acid (PAA) produced by reaction of triacetin (105 mM)and hydrogen peroxide (78 mM) at pH 6.5 in the presence of 1 mg/mL ofcommercial lipases. PAA PAA (ppm); (ppm); commercial lipase lipasesource 5 min 30 min no enzyme control 15 20 Meito MY Candida rugosa 2545 Meito SL Pseudomonas cepacia 0 0 Meito QLM Alcaligines sp. 35 85Amano F-DS Rhizopus oryzae 20 50 Amano M 10 Mucor javanicus 20 40 AmanoA 12 Aspergillus niger 70 140 BioCatalytics ICR 110 Candida antartica B55 110

EXAMPLE 29 B. subtilis ATCC31954™ Perhydrolase Activity with WettingAgents

A cell extract of E. coli UM2/pSW186 transformant expressing B. subtilisATCC 31954™ perhydrolase was prepared according to the proceduredescribed in Example 21. Separate 1-mL reactions containing triacetin(105 mM), hydrogen peroxide (78 mM), wetting agent COLATERIC® MSC-NA(mixed short chain sodium dipropionate; Colonial Chemical Co.),SURFYNOL® 2502 (an ethoxylated/propoxylated acetylenic-based surfactant;Air Products and Chemicals; Utrecht, NL), SURFYNOL® MD-20, SILWET® L7650(a polyalkyleneoxide modified polydimethylsiloxane; Chemtura Corp,Middlebury, Conn.) or SILWET® L8620; a siloxane-based surfactant), and 1mg of extract total protein in 50 mM phosphate buffer (pH 7.5) were runat 25° C. A control reaction was run by substituting 50 mM phosphatebuffer (pH 6.5) for the extract total protein solution to determine theconcentration of peracetic acid produced by chemical perhydrolysis oftriacetin by hydrogen peroxide in the absence of added extract protein.The concentration of peracetic acid in the reaction mixtures (Table 17)was determined according to the method of Karst et al. described inExample 2.

TABLE 17 Peracetic acid (PAA) produced by reaction of triacetin (105 mM)and hydrogen peroxide (78 mM) at pH 7.5 in the presence of 1 mg of totalextract protein/mL from transformant cell extracts of E. coli UM2/pSW186 expressing B. subtilis ATCC 31954 ™ perhydrolase. wetting totalPAA PAA wetting agent conc. protein (ppm); (ppm); agent (ppm) (mg/mL) 5min 30 min none 0 0 130 170 COLATERIC MSC-NA 1000 0 80 70 COLATERICMSC-NA 1000 1.0 745 1520 SURFYNOL ® 2502 1000 0 35 10 SURFYNOL ® 25021000 1.0 650 1210 SURFYNOL ® MD-20 1000 0 110 150 SURFYNOL ® MD-20 10001.0 555 1110 SILWET ® L7650 1000 0 50 0 SILWET ® L7650 1000 1.0 830 1360SILWET ® L8620 1000 0 60 135 SILWET ® L8620 1000 1.0 735 1145

EXAMPLE 30 B. subtilis BE1010 Perhydrolase Activity with Wetting Agents

A cell extract of E. coli UM2/pSW187 transformant expressing B. subtilisATCC 31954™ perhydrolase was prepared according to the proceduredescribed in Example 21. Separate 1-mL reactions containing triacetin(105 mM), hydrogen peroxide (78 mM), wetting agent (PLURONIC® 17R4 (apolyoxyalkylene ether surfactant; BASF, Mount Olive, N.J.), PLURONIC®L43 (a difunctional block copolymer surfactant), or SILWET® L7650), and1 mg of extract total protein in 50 mM phosphate buffer (pH 6.5) wererun at 25° C. A control reaction was run by substituting 50 mM phosphatebuffer (pH 6.5) for the extract total protein solution to determine theconcentration of peracetic acid produced by chemical perhydrolysis oftriacetin by hydrogen peroxide in the absence of added extract protein.The concentration of peracetic acid in the reaction mixtures (Table 18)was determined according to the method of Karst et al. described inExample 2.

TABLE 18 Peracetic acid (PAA) produced by reaction of triacetin (105 mM)and hydrogen peroxide (78 mM) at pH 6.5 in the presence of 1 mg of totalextract protein/mL from transformant cell extracts of E. coli UM2/pSW187 expressing B. subtilis BE1010 perhydrolase. wetting total PAA PAAwetting agent conc. protein (ppm); (ppm); agent (ppm) (mg/mL) 5 min 30min none 0 0 0 0 none 0 1.0 975 1345 PLURONIC ® 2500 0 0 0 17R4PLURONIC ® 17R4 2500 1.0 860 1360 PLURONIC ® L43 2500 0 0 0 PLURONIC ®L43 2500 1.0 855 1360 SILWET ® L7650 2500 0 0 0 SILWET ® L7650 2500 1.0975 1205

EXAMPLE 31 Perhydrolase Activity with Wetting and Chelating Agents

A cell extract of E. coli UM2/pSW187 transformant expressing B. subtilisBE1010 perhydrolase was prepared according to the procedure described inExample 21. Separate 1-mL reactions containing triacetin (105 mM),hydrogen peroxide (78 mM), wetting agent (SILWET® L7650), chelatingagent (TURPINAL® SL; etidronic acid; Solutia Inc., St. Louis, Mo.), and1 mg of extract total protein in 50 mM phosphate buffer (pH 6.5) wererun at 25° C. A control reaction was run by substituting 50 mM phosphatebuffer (pH 6.5) for the extract total protein solution to determine theconcentration of peracetic acid produced by chemical perhydrolysis oftriacetin by hydrogen peroxide in the absence of added extract protein.The concentration of peracetic acid in the reaction mixtures (Table 19)was determined according to the method of Karst et al. described inExample 2.

TABLE 19 Peracetic acid (PAA) produced by reaction of triacetin (105 mM)and hydrogen peroxide (78 mM) at pH 6.5 in the presence of 1 mg of totalextract protein/mL from transformant cell extracts of E. coli UM2/pSW187 expressing B. subtilis BE1010 perhydrolase. SILWET ® Turpinal ®total L7650 SL protein PAA (ppm); PAA (ppm); (ppm) (ppm) (mg/mL) 5 min30 min 0 0 0 21 50 1000 0 0 20 26 0 500 0 10 45 1000 500 0 0 100 0 0 1.01600 2245 1000 0 1.0 1550 2136 0 500 1.0 1520 2130 1000 500 1.0 15052080

EXAMPLE 32 Perhydrolase Activity with Wetting Agent, Chelating Agent andCorrosion Inhibitor

A cell extract of E. coil UM2/pSW187 transformant expressing B. subtilisBE1010 perhydrolase was prepared according to the procedure described inExample 21. Separate 1-mL reactions containing triacetin (105 mM),hydrogen peroxide (78 mM), wetting agent (SILWET® L7650), chelatingagent (TURPINAL® SL), corrosion inhibitor (benzotriazole) and 1 mg ofextract total protein in 50 mM phosphate buffer (pH 6.5) were run at 25°C. A control reaction was run by substituting 50 mM phosphate buffer (pH6.5) for the extract total protein solution to determine theconcentration of peracetic acid produced by chemical perhydrolysis oftriacetin by hydrogen peroxide in the absence of added extract protein.The concentration of peracetic acid in the reaction mixtures (Table 20)was determined according to the method of Karst et al. described inExample 2.

TABLE 20 Peracetic acid (PAA) produced by reaction of triacetin (105 mM)and hydrogen peroxide (78 mM) at pH 6.5 in the presence of 1 mg of totalextract protein/mL from transformant cell extracts of E. coli UM2/pSW187 expressing B. subtilis BE1010 perhydrolase. SILWET ® Turpinal ®total PAA PAA L7650 SL benzotriazole protein (ppm); (ppm); (ppm) (ppm)(ppm) (mg/mL) 5 min 30 min 0 0 0 0 0 0 0 0 0 1.0 795 1205 1000 500 10000 0 20 1000 500 1000 1.0 825 960 1000 500 2500 0 0 24 1000 500 2500 1.0795 960 1000 2000 2500 0 0 0 1000 2000 2500 1.0 270 450

EXAMPLE 33 Peracetic Acid Production using Immobilized B. subtilis ATCC31954™ or BE1010 Perhydrolase

A suspension of 0.50 g of AMBERZYME® Oxirane enzyme immobilizationpolymeric support (Rohm and Haas, Philadelphia, Pa.) in 5.0 mL of 0.225M sodium phosphate buffer (pH 8.0) containing 10 mg/mL of total solubleprotein from extracts (prepared as described in Example 21) of either E.coli KMP/pSW189 (expressing B. subtilis BE1010 perhydrolase) or E. colUM2/pSW186 (expressing B. subtilis ATCC 31954™ perhydrolase) was mixedon a rotating platform at room temperature for 24 h. The supernatant wasthen decanted from the immobilized enzyme, which was washed with four40-mL volumes of phosphate buffer (50 mM, pH 6.5) and stored at 5° C. inthis same buffer. The immobilized enzyme was dried by vacuum filtrationprior to use.

Separate 1-mL reactions containing triacetin (250 mM), hydrogen peroxide(1.0 M) and either 1.5 mg/mL or 5.0 mg/ml of immobilized perhydrolase(prepared as described above) in 50 mM phosphate buffer (pH 6.5) wererun at 25° C. A control reaction was run to determine the concentrationof peracetic acid produced by chemical perhydrolysis of triacetin byhydrogen peroxide in the absence of added immobilized enzyme. Theconcentration of peracetic acid in the reaction mixtures (Table 21) wasdetermined according to the method of Karst et al. described in Example2.

TABLE 21 Peracetic acid (PAA) produced by reaction of triacetin (250 mM)and hydrogen peroxide (1.0M) at pH 6.5 in the presence of immobilized B.subtilis ATCC 31954 ™ or BE1010 perhydrolase. PAA PAA catalyst loading(ppm); (ppm); immobilized perhydrolase (mg immob. enzyme/mL) 5 min 30min no enzyme 0 83 240 B. subtilis ATCC 31954 ™ 1.5 185 700 B. subtilisBE1010 1.5 502 1715 no enzyme 0 99 319 B. subtilis ATCC 31954 ™ 5.0 596972 B. subtilis BE1010 5.0 1669 2610

EXAMPLE 34 Perhydrolysis of a Mixture of Diacetin, Triacetin, andMonoacetin Using Perhydrolases from B. subtilis, B. licheniformis and C.thermocellum

Separate 1-mL reactions containing a mixture of diacetin (118 mM),triacetin (42 mM) and monoacetin (90 mM), hydrogen peroxide (1.0 M) and50 μg of extract total protein from an E. coli UM2 cell extract(prepared as described Example 21) that contained perhydrolase in 50 mMphosphate buffer (pH 6.5) were run at 25° C. A control reaction was runby substituting 50 mM phosphate buffer (pH 6.5) for the extract totalprotein solution to determine the concentration of peracetic acidproduced by chemical perhydrolysis of triacetin by hydrogen peroxide inthe absence of added extract protein. A second control reaction was runusing 50 μg of extract total protein prepared from an extract ofuntransformed E. coli UM2 to determine the background level of peracidproduced by the E. coli strain in the absence of expressed perhydrolase.The concentration of peracetic acid in the reaction mixtures (Table 22)was determined according to the method of Karst et al., described inExample 2.

TABLE 22 Peracetic acid (PAA) produced by reaction of a mixture ofdiacetin (118 mM), triacetin (42 mM) and monoacetin (90 mM) withhydrogen peroxide (1.0M) at pH 6.5 in the presence of 50 μg of totalextract protein/mL from transformant cell extracts of E. coli UM2expressing perhydrolase. PAA PAA transformant perhydrolase (ppm); (ppm);cell extract source 5 min 30 min no extract control 76 270 UM2 none(control) 110 276 UM2/pSW186 B. subtilis ATCC 31954 ™ 2352 4341UM2/pSW187 B. subtilis BE1010 2710 4713 UM2/pSW188 B. subtilis ATCC6633 ™ 2685 4234 UM2/pSW190 B. subtilis ATCC 29233 ™ 641 1889 UM2/pSW191B. licheniformis ATCC 14580 ™ 1183 2608 UM2/pSW193 C. thermocellum ATCC27405 ™ 1498 1708

EXAMPLE 35 Perhydrolysis of a Mixture of Diacetin, Triacetin, andMonoacetin Using Perhydrolase from B. subtilis BE1010

Separate 1-mL reactions containing a mixture of diacetin (49.6 mM),triacetin (17.6 mM) and monoacetin (37.8 mM), hydrogen peroxide (78 mM)and 1 mg or 2 mg of extract total protein from a cell extract (preparedas described Example 21) in 50 mM phosphate buffer (pH 6.5) were run at25° C. A control reaction was run by substituting 50 mM phosphate buffer(pH 6.5) for the extract total protein solution to determine theconcentration of peracetic acid produced by chemical perhydrolysis oftriacetin by hydrogen peroxide in the absence of added extract protein.The concentration of peracetic acid in the reaction mixtures (Table 23)was determined according to the method of Karst et al. described inExample 2.

TABLE 23 Peracetic acid (PAA) produced by reaction of triacetin (105 mM)and hydrogen peroxide (78 mM) at pH 6.5 in the presence of 1 mg or 2 mgof total extract protein/mL from transformant cell extracts of E. coliKLP18/pSW189 expressing B. subtilis BE1010 perhydrolase. total PAA PAAtransformant cell source of protein (ppm); (ppm); extract perhydrolase(mg/mL) PH 5 min 30 min no extract control 0 6.5 0 0 KLP18/pSW189 B.subtilis 1.0 6.5 475 423 BE1010 KLP18/pSW189 B. subtilis 2.0 6.5 505 463BE1010

EXAMPLE 36 Perhydrolysis of Acetylated Sugars by B. subtilis ATCC 31954™Perhydrolase

A cell extract of E. coli UM2/pSW186 transformant expressing B. subtilisATCC 31954™ perhydrolase was prepared according to the proceduredescribed in Example 21. Separate 1-mL reactions containing 0.1 Macetylated sugar (β-D-ribofuranose-1,2,3,5-tetraacetate,tri-O-acetyl-D-galactal, or tri-O-acetyl-D-glucal (Aldrich)), hydrogenperoxide (100 or 500 mM), 2 mg of extract total protein in 50 mMphosphate buffer (pH 6.5) were run at 25° C. A control reaction was runby substituting 50 mM phosphate buffer (pH 6.5) for the extract totalprotein solution to determine the concentration of peracetic acidproduced by chemical perhydrolysis of triacetin by hydrogen peroxide inthe absence of added extract protein. The concentration of peraceticacid in the reaction mixtures (Table 24) was determined according to themethod of Karst et al. described in Example 2.

TABLE 24 Peracetic acid (PAA) produced by reaction of acetylated sugar(100 mM) and hydrogen peroxide (100 or 500 mM) at pH 6.5 in the presenceof 2 mg of total extract protein/mL from transformant cell extracts ofE. coli UM2/pSW186 expressing B. subtilis ATCC 31954 ™ perhydrolase.hydrogen PAA PAA acetylated peroxide protein (ppm); (ppm); sugar (mM)(mg/mL) 5 min 30 min β-D-ribofuranose-1,2,3,5- 500 0 550 705tetraacetate β-D-ribofuranose-1,2,3,5- 500 2.0 1115 1540 tetraacetatetri-O-acetyl-D-galactal 500 0 220 225 tri-O-acetyl-D-galactal 500 2.0885 815 tri-O-acetyl-D-glucal 500 0 20 25 tri-O-acetyl-D-glucal 500 2.0420 275 β-D-ribofuranose-1,2,3,5- 100 0 52 37 tetraacetateβ-D-ribofuranose-1,2,3,5- 100 2.0 289 354 tetraacetatetri-O-acetyl-D-galactal 100 0 5 95 tri-O-acetyl-D-galactal 100 2.0 185175 tri-O-acetyl-D-glucal 100 0 65 0 tri-O-acetyl-D-glucal 100 2.0 10260

EXAMPLE 37 Perhydrolysis of Acetylated Sugars by B. subtilis BE1010Perhydrolase

A cell extract of E. coli KLP18/pSW189 transformant expressing B.subtilis BE1010 perhydrolase was prepared according to the proceduredescribed in Example 21. Separate 1-mL reactions containing 0.1 Macetylated sugar (β-D-ribofuranose-1,2,3,5-tetraacetate,tri-O-acetyl-D-galactal, or tri-O-acetyl-D-glucal (Aldrich)), hydrogenperoxide (100 or 500 mM), 2 mg of extract total protein in 50 mMphosphate buffer (pH 6.5) were run at 25° C. A control reaction was runby substituting 50 mM phosphate buffer (pH 6.5) for the extract totalprotein solution to determine the concentration of peracetic acidproduced by chemical perhydrolysis of triacetin by hydrogen peroxide inthe absence of added extract protein. The concentration of peraceticacid in the reaction mixtures (Table 25) was determined according to themethod of Karst et al. described in Example 2.

TABLE 25 Peracetic acid (PAA) produced by reaction of acetylated sugar(100 mM) and hydrogen peroxide (100 or 500 mM) at pH 6.5 in the presenceof 2 mg of total extract protein/mL from transformant cell extracts ofE. coli KLP18/pSW189 transformant expressing B. subtilis BE1010perhydrolase. hydrogen total PAA PAA acetylated peroxide protein (ppm);(ppm); sugar (mM) (mg/mL) 5 min 30 min β-D-ribofuranose-1,2,3,5- 500 0550 705 tetraacetate β-D-ribofuranose-1,2,3,5- 500 2.0 1465 1950tetraacetate tri-O-acetyl-D-galactal 500 0 185 375tri-O-acetyl-D-galactal 500 2.0 880 985 tri-O-acetyl-D-glucal 500 0 1040 tri-O-acetyl-D-glucal 500 2.0 770 405 β-D-ribofuranose-1,2,3,5- 100 052 37 tetraacetate β-D-ribofuranose-1,2,3,5- 100 2.0 360 437tetraacetate tri-O-acetyl-D-galactal 100 0 102 112tri-O-acetyl-D-galactal 100 2.0 305 262 tri-O-acetyl-D-glucal 100 0 1217 tri-O-acetyl-D-glucal 100 2.0 240 137

1. A formulation comprising: (a) an enzyme catalyst comprising anenzyme, wherein said enzyme has perhydrolase activity and comprises asignature motif that aligns with a reference sequence SEQ ID NO: 2 usingCLUSTALW, said signature motif comprising: (i) an RGQ motif at positionscorresponding to positions 118-120 of SEQ ID NO:2; (ii) a GXSQG motif atpositions corresponding to positions 179-183 of SEQ ID NO:2; and (iii)an HE motif at positions corresponding to positions 298-299 of SEQ IDNO:2; (b) a substrate selected from the group consisting of an ester, aglyceride, an acetylated saccharide, and a mixture thereof; and (c) asource of peroxygen.
 2. The formulation of claim 1, wherein thesubstrate is (i) an ester having the structure

wherein R₁═C1 to C7 straight chain or branched chain alkyl optionallysubstituted with an hydroxyl or a C1 to C4 alkoxy group and R₂═C1 to C10straight chain or branched chain alkyl, alkenyl, alkynyl, aryl,alkylaryl, alkylheteroaryl, heteroaryl, (CH₂CH₂—O)_(n)H or(CH₂CH(CH₃)—O)_(n)H and n=1 to 10; (ii) a glyceride having the structure

wherein R₁═C1 to C7 straight chain or branched chain alkyl optionallysubstituted with an hydroxyl or a C1 to C4 alkoxy group and R₃ and R₄are individually H or R₁C(O); (iii) an acetylated saccharide selectedfrom the group consisting of acetylated monosaccharides, acetylateddisaccharides, and acetylated polysaccharides; or (iv) a mixturethereof.
 3. The formulation of claim 2, wherein the substrate isselected from the group consisting of methyl lactate, ethyl lactate,methyl glycolate, ethyl glycolate, methyl methoxyacetate, ethylmethoxyacetate, methyl 3-hydroxybutyrate, ethyl 3-hydroxybutyrate,monoacetin, diacetin, triacetin, monopropionin, dipropionin,tripropionin, monobutyrin, dibutyrin, tributyrin, glucose pentaacetate,xylose tetraacetate, acetylated xylan,β-D-ribofuranose-1,2,3,5-tetraacetate, tri-O-acetyl-D-galactal,tri-O-acetyl-glucal, and mixtures thereof.
 4. The formulation of claim1, wherein the signature motif of the enzyme further comprises an LXDmotif at positions corresponding to positions 267-269 of SEQ ID NO:2using CLUSTALW.
 5. The formulation of claim 4, wherein the signaturemotif is selected from the group consisting of SEQ ID NO:61 and an aminoacid sequence at least 50% identical to SEQ ID NO:61.
 6. The formulationof claim 1, which produces a peroxycarboxylic acid selected from thegroup consisting of peracetic acid, perpropionic acid, perbutyric acid,perlactic acid, perglycolic acid, permethoxyacetic acid,per-β-hydroxybutyric acid, and mixtures thereof.
 7. The formulation ofclaim 6, wherein the peroxycarboxylic acid produced is peracetic acid.8. The formulation of claim 1 further comprising water.